Compositions and methods for the treatment of addiction and other neuropsychiatric disorders

ABSTRACT

The present invention is based, in part, on our discovery that certain types of therapeutic agents can be used in combination to treat a variety of neuropsychiatric and related disorders, including addiction (e.g., to a substance or to an activity) as well as to alleviate some of the symptoms experienced during menopause or associated with the menstrual cycle. Regardless of the precise formulation, the compositions of the invention can include at least one active ingredient that targets the hypothalamo-pituitary-adrenal (HPA) axis and at least one active ingredient that targets the prefrontal cortex. Either or both of these types of agents can be combined with an agent that inhibits activity in the sympathetic nervous system. Thus, the compositions or combination pharmacotherapies can also include an agent that inhibits a beta-adrenergic receptor or that otherwise acts as an anti-hypertensive or anxiolytic agent.

RELATED APPLICATIONS

This application claims the benefit of U.S. Application No. 60/735,507,filed Nov. 10, 2005, and U.S. Application No. 60/764,727, filed Feb. 2,2006. For the purpose of any U.S. patent that may issue from the presentapplication, the contents of these two earlier-filed applications arehereby incorporated by reference herein in their entireties.

TECHNICAL FIELD

This invention relates to methods for treating a variety of conditionsand disorders, including neuropsychiatric disorders such as addiction,anxiety, depression, schizophrenia, and related conditions (e.g.,insomnia), and more generally to methods of making and usingpharmaceutical formulations that target distinct tissues within thenervous and endocrine systems.

SUMMARY OF THE INVENTION

The present invention is based, in part, on our discovery that certaintypes of therapeutic agents can be used in combination to treat avariety of neuropsychiatric and related disorders, including addiction(e.g., an addiction to a substance such as a drug or to an activity suchas gambling). More specifically, these agents can be used to treateating disorders; depression; disruptive behavior disorders (e.g.,attention deficit disorders such as attention deficit and hyperactivitydisorder (ADHD)); schizophrenia; anxiety (e.g., anxiety experienced inthe context of post-traumatic stress disorder); sleep disorders; and/orrelated or resulting conditions. For example, the invention includescompositions and methods that can be used to treat or prevent obesity orvarious eating disorders. The compositions can also be used in thetreatment or prevention of insomnia, which can occur independently or inconnection with conditions associated with stress or stress-relateddisorders (e.g., an anxiety). More generally, these conditions can bedescribed as those associated with hypercortisolism, other activitieswithin the hypothalamic-pituitary-adrenal (HPA) axis (e.g., alteredregulation of adrenocorticotropic hormone (ACTH)) or prefrontal cortex,and/or excessive activity in the sympathetic nervous system.

The present compositions can also be used to alleviate some of thesymptoms experienced during menopause or associated with the menstrualcycle (e.g., pre-menstrual syndrome (PMS)).

Accordingly, the invention features pharmaceutical compositions andmethods by which they can be prepared and administered (e.g., prescribedand self-administered) to a patient. Given the current norms, we expectthe therapeutic agents described herein will be formulated in a singlepreparation (e.g., a single tablet, capsule, or the like, which may bedesigned to produce a sustained and controlled release) and administeredorally. The invention is not so limited, however, and exemplaryalternatives for combining and administering the therapeutic agents aredescribed further below (e.g., solutions can be administeredintravenously).

Regardless of the precise formulation or configuration, the compositionscan include at least one active ingredient that targets thehypothalamo-pituitary-adrenal (HPA) axis and at least one activeingredient that targets the prefrontal cortex (e.g., by targetingGABA_(A) receptors in the prefrontal cortex). More specifically, thecompositions can include at least one of a first active agent that:inhibits corticotropin-releasing hormone (CRH); inhibitsadrenocorticotropic hormone (ACTH); and/or inhibits cortisol. Forexample, the agent can reduce the ability of CRH to stimulate therelease of ACTH from the pituitary gland, reduce the ability of ACTH tostimulate the release of cortisol from the adrenal gland, or inhibitcortisol synthesis, secretion, or activity. For example, while thepresent compositions are not limited to those that exert their effect byany particular mechanism, agents that inhibit cortisol activity may doso by competing with cortisol for glucocorticoid receptor binding and/orblocking a downstream event such as receptor activation, dimerization ortranscriptional signaling through a glucocorticoid response element.These agents may also be agents that bind to another type ofadrenocorticosteroid receptor, such as a mineralocorticoid receptor,and/or that inhibit downstream events following mineralocorticoidreceptor binding.

The compositions can further include at least one of a second activeagent that targets the prefrontal cortex by, for example, increasing theexpression or activity of gamma-aminobutyric acid (GABA); mimickingGABA; inhibiting GABA metabolism in the prefrontal cortex; and/orotherwise stimulating GABA signaling in the prefrontal cortex. As noted,the compositions can contain these first and second agents by virtue ofa physical combination of the agents per se (e.g., in an admixture orsuspension) in, for example, a sustained-release preparation. In otherembodiments, the compositions can be combined by virtue of a sharedpackaging (e.g., tablets containing the first active agent and tabletscontaining the second active agent can be combined in a single blisterpack, optionally marked to indicate days of the week and/or times of theday). Solutions for intravenous administration can similarly bepackaged, with one solution containing the first agent and one solutioncontaining the second agent, with instructions for simultaneous orsequential administration.

In specific embodiments, the compositions can include one or more of thetypes of agents listed in the first column of the following table andone or more of the types of agents listed in the second column.

First active agent Second active agent An agent that inhibits CRH in theAn agent that directly or indirectly HPA axis or CNS, including thestimulates GABA in the prefrontal prefrontal cortex cortex An agent thatinhibits ACTH in An agent that mimics GABA in the the pituitary glandprefrontal cortex An agent that inhibits cortisol in An agent thatinhibits GABA the adrenal gland metabolism

Either or both of these types of agents can be combined with an agentthat inhibits activity in the sympathetic nervous system (e.g., abeta-blocker such as propranolol (Inderal®)). Beta-blockers and otheragents (e.g., anxiolytics) that can be included as a “third” agent aredescribed further below. Thus, the compositions or combinationpharmacotherapies can include an agent that inhibits a beta-adrenergicreceptor (e.g., by binding the receptor and inhibiting its interactionwith epinephrine) or that otherwise act as anti-hypertensives oranxiolytic agents.

In specific embodiments, an agent that inhibits CRH can be combined withan agent that stimulates GABA in the prefrontal cortex and an agent thatinhibits activity in the sympathetic nervous system; an agent thatinhibits ACTH can be combined with an agent that stimulates GABA in theprefrontal cortex and an agent that inhibits activity in the sympatheticnervous system; an agent that inhibits cortisol can be combined with anagent that stimulates GABA in the prefrontal cortex and an agent thatinhibits activity in the sympathetic nervous system; and one or moreagents that bind to adrenocorticosteroid receptors can be combined withan agent that inhibits activity in the sympathetic nervous system. Inany of these exemplary embodiments, the referenced agent can be onedescribed herein (e.g., an agent that stimulates GABA can be an agentthat directly or indirectly stimulates GABA in the prefrontal cortex; anagent that mimics GABA in the prefrontal cortex (e.g., a GABA receptor(e.g., GABA_(A)) agonist); or an agent that inhibits GABA metabolism).

GABA is an inhibitory neurotransmitter that hyperpolarizes the inhibitedneuron following receptor binding. This binding opens chloride andpotassium channels, either directly or indirectly. Activated ionotropicreceptors are ion channels themselves while the metabotropic receptorsare G protein-coupled receptors that activate ion channels via theintermediary G proteins. Either type of receptor can be activated by anagent serving to mimic GABA and thereby target the prefrontal cortex:Other agents can act by increasing GABA synthesis. For example, nucleicacids encoding the synthetic enzyme L-glutamic acid decarboxylase, or abiologically active fragment or other mutant thereof, can beadministered to a patient who is likely to benefit from the methodsdescribed herein (e.g., a patient who has demonstrated or who has beendiagnosed as having an addiction (other patients amenable to treatmentare described elsewhere herein))

In other embodiments, the therapeutic composition can be a combinationof at least two or three of (e.g., two, three, or four of): an agentthat inhibits CRH, an agent that inhibits ACTH, an agent that inhibitscortisol (or binds an adrenocorticoid receptor), an agent that directlyor indirectly stimulates GABA in the prefrontal cortex, an agent thatmimics GABA in the prefrontal cortex, or an agent that inhibits GABAmetabolism and an agent that inhibits activity in the sympatheticnervous system.

Unless the context indicates otherwise, we use the term “agent” tobroadly refer to any substance that affects a target molecule (e.g., aligand or the receptor to which it binds) or a target region of thebrain or endocrine system in a clinically beneficial way (e.g., toinhibit HPA axis activation following a patient's exposure to one ormore conditioned environmental cues). For example, we may refer tochemical compounds such as metyrapone (Metopirone®) as “agents”. We mayalso use the term “compound” to refer to conventional chemical compounds(e.g., small organic or inorganic molecules). The “agent” may also be aprotein or protein-based molecule, such as a mutant ligand or antibody.Other useful agents include nucleic acids or nucleic acid-based entitiessuch as antisense oligonucleotides or RNA molecules that mediate RNAiand the vectors used for their delivery. For example, we may refer to anantibody that specifically binds and alters (e.g., inhibits) theactivity of CRH (e.g., a human or humanized anti-CRH antibody) or to anucleic acid (e.g., an siRNA or shRNA) that specifically interacts with,and inhibits translation of, an RNA encoding CRH as an “agent” thatinhibits CRH. CRH is only one of the molecules that can be targeted;ACTH, cortisol, and GABA can be targeted by any of the types of agentsdiscussed herein in reference to CHR. Compounds useful in the inventioninclude those that bind a cortisol receptor. Preliminary resultsindicate that corticosterone is elevated in an animal model ofaddiction.

While agents useful in the compositions of the invention are describedfurther below, we note here that agents that can inhibit CRH in the HPAinclude agents (e.g., nucleic acids) that inhibit CRH expression; agentsthat inhibit CRH production or secretion by way of participation in anegative feedback loop; antibodies that specifically bind to and inhibitCRH; CRH receptor antagonists (e.g., proteins, including antibodies,that bind the CRH receptor and inhibit signal transduction or that actintracellularly to inhibit the second messengers normally generated inresponse to CRH receptor binding); chemical compounds (e.g., smallmolecules) that inhibit the expression, secretion, or activity of CRH orthe CRH receptor (e.g., compounds that inhibit the ability of CRH tobind cognate receptors in the pituitary); and agents that facilitate CRHmetabolism. As noted, other agents can inhibit ACTH. For example, thecompositions of the invention can include agents (e.g., nucleic acids)that inhibit ACTH expression; agents that inhibit ACTH production orsecretion by way of participation in a negative feedback loop;antibodies that specifically bind to and inhibit ACTH; ACTH receptorantagonists (e.g., proteins that bind the ACTH receptor and inhibitsignal transduction or that act intracellularly to inhibit the secondmessengers normally generated in response to ACTH receptor binding);chemical compounds that inhibit the expression, secretion, or activityof ACTH or the ACTH receptor (e.g., compounds that inhibit the abilityof ACTH to bind cognate receptors in the adrenal gland); and agents thatfacilitate ACTH metabolism.

Agents that inhibit CRH include [Met18, Lys23, Glu27,29,40, Ala32,41,Leu33,36,38] CRF9-41, which is abbreviated as alpha-helical CRF(9-41)and has the sequenceAsp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Met-Leu-Glu-Met-Ala-Lys-Ala-Glu-Gln-Glu-Ala-Glu-Gln-Ala-Ala-Leu-Asn-Arg-Leu-Leu-Leu-Glu-Glu-Ala(SEQ ID NO:1)) and biologically active fragments or variants thereof(Rivier et al., Science 224:889, 1984). Another agent that inhibits CRHis [D-Phe12, Nle21,38, (αMeLeu37)] CRF(12-41), which is abbreviated asD-Phe CRF12-41, and biologically active fragments and variants thereof.Other agents that inhibit CRH include Astressin®; CP-154,526; NB127914,Antalarmin®; CRA1000; CRA1001, and Antisauvagine-30. See also U.S. Pat.Nos. 6,326,463; 6,323,312; 4,594,329, and 4,605,642. It is known in theart that deleting certain N-terminal amino acid residues from CRFproduces CRF antagonists, and these antagonists (e.g., CRF(8-41),CRF(9-41), and CRF(10-41)) can be used in the present compositions andmethods. Cyclic peptides that inhibit CRF are described in U.S. Pat. No.6,323,312 and can be used in the present compositions and methods.

To inhibit ACTH, one can administer a sufficient amount of ACTH toinhibit ACTH through feedback inhibition or to down-regulate the ACTHreceptor.

Chemical compounds that inhibit cortisol include metyrapone,ketoconazole, and aminoglutethamide. Useful compounds and other agents,including those described with particularity herein and/or otherwiseknown in the art, can act at any point along the HPA axis todown-regulate the effect of cortisol (i.e., they can act on the target(e.g., cortisol) directly (e.g., by binding to and inhibiting thetarget) or indirectly (e.g., by inhibiting a molecule active upstreamfrom the target in the HPA axis)).

Substance P antagonists and vasopressin inhibitors can also be used inthe present compositions and methods to inhibit activity within the HPAaxis. Substance P is an 11-amino acid neuropeptide that binds aneurokinin 1 receptor. Antagonists include Aprepitant®, which iscurrently available for chemotherapy-induced nausea, and MK-0869, whichis an antidepressant and substance P receptor antagonist.[D-Arg¹,D-Pro²,D-Trp^(7,9),Leu¹¹]SP has been administered intravenouslyas a substance P antagonist.

Agents that augment endocannabinoid signaling can also be used toinhibit activity in the HPA axis and are useful in the presentcompositions and methods. These agents may stimulate the expression oractivity of an endocannabinoid or may, for example, be or mimic anendocannabinoid (see Patel et al., Endocrinol. 145:5431-5438, 2004).While the invention is not limited to agents that exert their positiveeffect on the disorders and other conditions described herein by anyparticular mechanism, we note that endocannabinoids can inhibit therelease of vasopressin from the posterior pituitary (Tasker, Endocrinol.145:5429-5430, 2004). 29-5430. Exogenous cannabinoids have been shown tostimulate the HPA, but at least one such compound, CP55940, can insteadreduce the stress-induced secretion of HPA hormones (Thomas et al., J.Pharmacol. Exp. Ther. 285:285-292, 1998).

An agent that directly or indirectly stimulates GABA in the prefrontalcortex may do so by directly or indirectly increasing the synthesis,release, or activity of GABA. Activity can be enhanced, for example, byenhancing the interaction between GABA and a cognate receptor. There arevarious ways to enhance this interaction, including increasing theconcentration of GABA, providing a receptor agonist, or altering thekinetics of receptor binding and signal transduction. GABA concentrationcan, in turn, be increased by increasing GABA synthesis or inhibitingGABA metabolism. GABA concentrations are, in effect, also increased bythe administration of agents that mimic GABA. With respect to indirectstimulation, any agent (e.g., an antidepressant) that preferentiallyincreases dopaminergic or noradrenergic activity in the prefrontalcortex can indirectly affect (i.e., stimulate) GABA in the prefrontalcortex. Mirtazapine is an example of an antidepressant agent that couldbe used to indirectly stimulate GABA; atomoxetine is an example ofanother type of agent that can be similarly used. Gabapentin(Neurontin™) is an example of an agent that mimics the effect of GABA,and direct stimulators include any benzodiazepine (e.g., oxazepam((Serax®) or chlordiazepoxide) or alprazolam (Xanax®). Other usefulagents such as muscimol and baclofen may stimulate GABA through theGABA_(A) or GABA_(B) receptor, respectively. Other GABA agonists ormimics include progabide, riluzole, baclofen, vigabatrin, valproic acid(Depakote™), tiagabine (Gabitril™), lamotrigine (Lamictal™), phenytoin(Dilantin™), carbamazepine (Tegretol™) and topiramate (Topamax™).

While dosages are described further below, we note here that, whenagents used within the compositions of the invention are ones that arepresently known and used to treat patients, the dosage of at least oneof the agents required in the context of our combination therapy may beless than the dosage at which that agent is currently and typicallyprescribed. For example, where the present compositions include abenzodiazepine that is currently used in the treatment of anxiety, theamount of that compound administered to a patient for the treatment ofaddiction can be less than a physician would have typically prescribedfor the treatment of anxiety. In some instances, the dosages of both ofthe agents within the present compositions will be less than thetraditional dosages of those agents. While the compositions of theinvention are not limited to those that have particular advantages, webelieve the ability to use low-dose formulations will reduce theincidence of side effects as well as the abuse potential associated withsome of the agents. It is understood in the art that some patients maybe more or less sensitive to a particular dosage of a given medication.In the present case, as is generally true, patients and their healthcare providers can monitor treatment for a desired effect and dosagesmay be variously adjusted (e.g., over time).

The amounts of chemical compounds within the present compositions canvary. For example, a patient may receive from about 1-1000 mg of a givenfirst agent and 1-1000 mg of a given second agent at defined intervals.Where a third agent is included, the formulation can include and thepatient may receive from 1-1000 mg of the third agent. For example, thepatient can be treated every so-many hours (e.g., about every 2, 4, 6,8, 12, or 24 hours), every so-many days (e.g., once a day, once everyother day, once every three days), or every so-many weeks (e.g., once aweek). For example, a patient may receive at least or about 5-1500 mg(e.g., at least or about 5, 10, 25, 50, 100, 200, 250, 300, 400, 450,500, 550, 600, 650, 700, 750, 800, 850, 900, 1000, 1250, or 1500 mg)) ofa first agent and at least or about 5-500 mg (e.g., at least or about 1,5, 10, 20, 25, 30, 35, 40, 45, 50, 100, 200, 250, 300, 400, 450, 500) ofa second agent from 1-4 times per day. Under such a regime, a patientcould receive at least or about 10-6000 mg of a first agent (e.g., atleast or about 25-1500 mg; 50-1250 mg; 100-1250 mg; 100-1000 mg;250-1000 mg; 500-1000 mg; 750-1000 mg (e.g., about 750 mg or about 1000mg)) such as metyrapone or ketoconazole. Under either the same or adifferent regime, a patient could receive about 5-100 mg of a secondagent (e.g., about 5-50 mg; about 5-40 mg; about 5-30 mg; about 5-20 mg;about 5-10 mg; about 10-50 mg; about 10-40 mg; about 10-30 mg; about20-50 mg; about 20-40 mg; about 20-30 mg; about 30-50 mg; or about 30-40mg of a first agent). As noted, the second agent can be abenzodiazepine, such as oxazepam. As noted, appropriate dosages can bedelivered over time from a sustained-release formulation, which may beadministered at daily or weekly intervals. Where particular formulationsor devices are used (e.g., an infusion pump), administration may proceedwithout the need for patient intervention for longer periods of time.

The amounts of the agents within a pharmaceutical preparation may be thesame or different (e.g., the ratio of the first agent to the second canbe at least or about 100:1; 90:1; 80:1; 75:1; 70:1; 65:1; 60:1; 55:1;50:1; 45:1; 40:1; 35:1; 30:1; 25:1; 20:1; 15:1; 10:1; 9:1; 8:1; 7:1;6:1; 5:1; 4:1; 3:1; 2:1; or about 1:1). For example, a composition cancontain about 1 equivalent of oxazepam to about 25-50 equivalents ofmetyrapone; about 25-50 equivalents of ketoconazole to about 1equivalent of alprazolam; about 25-50 equivalents of ketoconazole toabout 1 equivalent of oxazepam; about 25-50 equivalent of metyrapone toabout 1 equivalent of alprazolam; about 1 equivalent of muscimol toabout 25-50 equivalents of CP-154,526; or about 1 equivalent of muscimolto about 25-50 equivalents of metyrapone. An equivalent can be a unit ofweight (e.g., a milligram). The ratios can run differently, however,with the amount of the second agent exceeding the amount of the firstagent (by, for example, the varying extent described here). The relativeamounts of the active ingredients can also be expressed in terms ofpercentage. For example, relative to one another, the amount of thesecond agent can be at least or about 1-99% of the amount of the secondagent. Where a third agent is included to inhibit the sympatheticnervous system, the relative amount of that agent can also vary withrespect to the first and second agents. For example, relative to oneanother, the amount of the third agent can be at least or about 1-99% ofthe amount of the first or second agent. Where the third agent isincluded in a composition and/or used in a treatment regime, it mayallow use of either the first and/or the second agent in an amount thatis lower than predicted or that is required for efficacy in the absenceof the third agent.

The pharmaceutical compositions, which are described further below, caninclude standard ingredients such as carriers and preservatives. Thecompositions can also include substances (e.g., a polyethylene glycol)to increase the solubility of the active ingredients. Typically, theactive ingredients will account for a minority of the overallcomposition. For example, the first, second, and/or third agents canconstitute about 1-50% of the pharmaceutical composition (e.g., about1-40%; 1-30%; 1-20%; 1-10%; 2-40%; 2-30%; 2-20%; 2-10%; 2-5%; 3-40%;3-30%; 3-20%; 3-10%; 3-5%; 4-40%; 4-30%; 4-20%; 4-10%; 4-5%; 1-2%; 1-3%;1-4%; 2-4%; 2-3%; or 3-4% of the pharmaceutical composition).

When an agent “targets” an area within a patient's nervous system orendocrine system, it affects the activity of cells within that area insuch a way as to confer a benefit on the patient. For example, where apatient is addicted to a substance or activity, the benefit can be areduction in the patient's engagement with that substance or activity.For example, the patient may use the substance or carry out the activityless frequently or to a lesser extent than one would expect in theabsence of treatment or to a lesser extent than prior to treatment.Thus, the benefit can be characterized as a reduction in the risk ofrelapse, even in the presence of conditioned environmental cues. Theclinical benefit can be subjective in that patients may report areduction in their craving for a substance or activity. Thus, thecompounds and methods of the invention can be used to promote abstinenceor periods of abstinence that are longer than one would expect in theabsence of treatment. Achieving any detectable improvement constitutes“treatment” of an addiction with the present compositions and methods;complete recovery may be achieved, but is not required to constitutetreatment. The same is true regarding other indications. For example, adetectable improvement in the event of an anxiety-associated disorder,an eating disorder, a sleeping disorder, schizophrenia, or an unpleasantsymptom of menopause constitutes treatment. Complete absence of anydifficulty is not required.

While we believe we understand certain events that occur in the courseof treatment, the compositions of the present invention are not limitedto those that work by affecting any particular cellular mechanism. Ourworking hypothesis is that, with respect to addiction, the cues thattrigger relapse to undesirable behaviors (e.g., addictive behaviors)produce those behaviors (or desires to behave) through a conditionedactivation of the HPA axis that affects neuronal activity in theprefrontal cortex. More specifically, the conditioned activation of theHPA axis increases the secretion of corticotropin-releasing hormone(CRH), adrenocorticotropic hormone (ACTH) and cortisol (corticosteronein rats) and these hormones, in turn, affect activity in the prefrontalcortex (the medial prefrontal cortex in rats), a brain region involvedin reward, judgment and other activities related to a propensity torelapse. When administered to treat an addiction, we believe thecombination therapies described herein reduce the likelihood of relapseby decreasing activity within the HPA axis and/or the prefrontal cortex.This reduces the cue-induced secretion of CRH, ACTH, and/or cortisol tolevels too low to evoke the cravings associated with addiction to asubstance or undesirable behavior. As these hormones (CRH, ACTH, andcortisol) affect activity in the prefrontal cortex, we expect prefrontalactivity to subsequently decline as well, and the second agent of thecomposition(s) can facilitate that decline.

As noted, the active ingredients of the present compositions can becombined in a single formulation or by virtue of their packaging.Accordingly, the present invention features kits containing singleformulations and/or dual-packaged formulations together withinstructions for their use. For example, the compositions can either becombined within a single tablet or capsule or divided between tabletsand placed within a blister pack optionally marked to indicate the dayor time of day it should be taken.

As noted, the compositions can be used to treat addiction to a varietyof compounds (i.e., to treat substance abuse) or activities. Forexample, the compositions can be used to treat addiction to stimulants(e.g., cocaine, amphetamines, methamphetamines, methylphenidate, andrelated stimulants), opiates (e.g., heroin, codeine, hydrocodone, andrelated opioid drugs), nicotine, alcohol, prescription medications(e.g., medications prescribed for pain management such as Percodan® orPercocet®), and naturally-occurring plant-derived drugs (e.g. marijuana,tobacco, and the addictive agents therein). Patients being treated withmethadone are also candidates for treatment with the compositionsdescribed herein. The present compositions may help such patientsstep-down and discontinue use of methadone. Patients who engage inaddictive behaviors can also be identified and treated. These patientsmay be suffering from an addiction to gambling, sex, or food. In each ofthese disorders, conditioned cues are believed to induce or contributeto relapse.

Alternatively, or in addition, the compositions described herein can beused to treat other neuropsychiatric disorders that involve HPA axisactivity and the prefrontal cortex. These include anxiety, including butnot limited to anxiety associated with panic disorder, obsessivecompulsive disorder (OCD), post-traumatic stress disorder (PTSD), socialanxiety disorder, generalized anxiety disorder, and obesity. Patientsdiagnosed as suffering from depression can also be treated. Theirdepression can be, but is not necessarily, associated with majordepressive disorder, dysthymia, bipolar depression, depressionassociated with medical conditions, and depression associated withsubstance abuse.

Other conditions amenable to treatment are obesity and various eatingdisorders, including Prader Willi Syndrome. Other patients amenable totreatment include those suffering from schizophrenia; those withdisruptive behavior disorders (e.g., attention-deficit disorder (ADD) orADHD); those experiencing menopause; and those suffering from amenstrual cycle-related syndrome (e.g., PMS). Other conditions amenableto treatment are insomnia and various sleep disorders.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A and 1B are bar graphs illustrating the effect of thecombination of metyrapone and oxazepam on intravenous cocaineself-administration in rats. The number of cocaine infusions is plottedin FIG. 1A, and the number of infusions expressed as a percentage of thebaseline is plotted in FIG. 1B.

FIGS. 2A and 2B are bar graphs illustrating the effect of thecombination of metyrapone and oxazepam on intravenousself-administration of three different doses of cocaine in rats. Thenumber of infusions per session is plotted in FIG. 1A, and the sameresult, expressed as a percentage of the base, is plotted in FIG. 2B.

FIG. 3 is a bar graph illustrating the effect of the combination ofketoconazole and alprazolam on intravenous cocaine self-administrationin rats. The number of infusions is plotted.

FIG. 4 is a bar graph illustrating the effect of the combination ofketoconazole and alprazolam on intravenous self-administration of threedifferent doses of cocaine in rats.

FIG. 5 is a bar graph illustrating the effect of the combination ofketoconazole and oxazepam on intravenous cocaine self-administration inrats. The infusion are expressed as a percentage of baseline.

FIG. 6 is a bar graph illustrating the effect of the combination ofCP-154,526 and oxazepam on intravenous cocaine self-administration inrats. The infusions are expressed as a percentage of baseline.

FIG. 7 is a bar graph illustrating the effect of the combination ofmetyrapone and alprazolam on intravenous cocaine self-administration inrats.

FIG. 8 is a bar graph illustrating the effect of the combination ofmuscimol and CP-154,526 on intravenous cocaine self-administration inrats. The infusions are expressed as a percentage of baseline.

FIG. 9 is a bar graph illustrating the effect of the combination ofmuscimol and metyrapone on intravenous cocaine self-administration inrats. The infusions are expressed as a percentage of baseline.

FIG. 10 is a bar graph illustrating the effect of the combination ofmetyrapone and oxazepam on the cue-induced reinstatement of extinguishedcocaine-seeking behavior in rats.

FIG. 11 is a bar graph illustrating the effect of chronic injections ofmetyrapone on the cue-induced reinstatement of extinguishedcocaine-seeking behavior in rats.

FIG. 12 is a bar graph illustrating the effect of CP-154,526 andoxazepam on the cue-induced reinstatement of extinguishedcocaine-seeking behavior in rats.

FIG. 13 is a schematic representing a synthetic pathway for synthesis ofmetyrapone.

FIG. 14 is a table summarizing the test conditions and results of apharmacokinetic analysis of cocaine, metyrapone, and oxazepam.

DETAILED DESCRIPTION

The compositions described herein include two or more therapeutic agentsfor the treatment of addiction, other neuropsychiatric disorders, andindependent or associated conditions. One or more of the agents includedin the formulations can be an agent that is currently available but notcurrently prescribed for the indication(s) described herein. Forexample, metyrapone is commonly used to diagnose malfunction of theadrenal glands, and oxazepam is a benzodiazepine used to treat anxietyand related disorders. Both of these drugs affect physiological systemsrelated to stress and the subsequent activation of the HPA axis.Alternatively, one or more of the agents can be newly formed inaccordance with the teachings herein. For example, an antisenseoligonucleotide or an RNA molecule that mediates RNAi can be producedgiven the sequence(s) of the target(s) discovered (i.e., CRH, ACTH, aGABA receptor (e.g., GABA_(A) or a component of the GABA_(A) receptorcomplex, as can be targeted by any of the “second” agents describedherein) or β adrenergic receptors in the sympathetic nervous system. Thesequences of these targets are known or readily available to one ofordinary skill in the art, as are methods for making antisenseoligonucleotides and RNA molecules that mediate RNAi. Other usefulagents, whether previously available or newly made, include antibodiesthat specifically bind a ligand identified herein (e.g., CRH, ACTH, orGABA) or a receptor activated in response to conditioned environmentalcues (e.g., a receptor for CRH, ACTH, cortisol, or GABA). Where an agentis employed to inhibit activity in the sympathetic nervous system, itmay be a chemical compound, such as those provided herein, or anothertype of agent. For example, one can administer nucleic acids or nucleicacid-based agents to inhibit the expression of β adrenergic receptors orantibodies that specifically bind and antagonize these receptors. Uponspecific binding, the antibody can act as an agonist or antagonist ofthe entity bound, as desired to facilitate or inhibit cellular activitymediated by receptor binding. For example, an antibody that specificallybinds CRH can act as a CRH antagonist; an antibody that specificallybinds a GABA receptor can act as a GABA receptor agonist; an antibodythat specifically binds a β adrenergic receptor can act as an adrenalineantagonist; an antibody that specifically binds a glucocorticoidreceptor can act as an antagonist to inhibit cortisol; and so forth.

Work conducted in our laboratory has demonstrated that the HPA axisplays an important role in drug addiction (Goeders,Psychoneuroendocrinology 22:237, 1997; Goeders, J. Pharmacol. Exp. Ther.301:785-789, 2002; Goeders, Psychoneuroendocrinology 27:13-33, 2002;Goeders, Eur. Neuropsychopharmacology 3:435-441, 2003), and we now havedata indicating that certain combinations of drugs (e.g., thecombination of metyrapone and oxazepam) are effective in treatingaddiction (as evidenced by reducing cocaine reward). Accordingly, theinvention features compositions that represent combined therapeuticagents (e.g., combinations of two or three agents that target theregions of the nervous and/or endocrine systems (e.g., the HPA axis andthe sympathetic nervous system) described herein) and methods oftreating patients with these agents (e.g., with a “first” and “second”agent or a “first” and “third” agent, as described herein).

Regardless of the substance or activity to which a patient is addicted,the extent of the addiction can vary; it may, to a greater or lesserextent impact the patient's ability to participate in or cope withlife's daily events, and it may recur with varying frequency (e.g., thepatient may experience a rare relapse or a fairly regular and/orfrequent relapse).

The agents can be categorized in various ways, and the compositions ofthe invention can include two or more agents of the same or differenttypes. For example, the agents can be categorized as chemical compounds(e.g., metyrapone and topiramate); as protein or protein-basedmolecules, such as mutant ligands (e.g., a ligand that binds but doesnot activate or fully activate its cognate receptor) as antibodies; oras nucleic acids or nucleic acid-based entities, such as antisenseoligonucleotides or RNA molecules that mediate RNAi. Thus, thecompositions of the invention can include two or more chemicalcompounds; two or more distinct protein or protein-based molecules; ortwo or more distinct nucleic acids or nucleic acid-based entities.Alternatively, the compositions can include two different types ofagents (e.g., a protein and a nucleic acid or a chemical compound and aprotein such as an antibody or an active fragment thereof). The methodsby which patients are treated can similarly include administration oftwo or more chemical compounds; two or more distinct proteins orprotein-based molecules; two or more distinct nucleic acids or nucleicacid-based entities; or any combination of agents of these various types(e.g., a protein and a nucleic acid).

Either or both of the agent(s) that target(s) the HPA axis and theagent(s) that target(s) the prefrontal cortex can be combined with anagent that inhibits activity in the sympathetic nervous system. Eitheror both of these types of agents can be combined with a beta blocker,suitable examples of which are provided below, or another type ofantihypertensive and/or anxiolytic agent (e.g., an angiotensin IIinhibitor such as candasartan). The third agent (i.e., the agent used inaddition to the agent that targets the HPA axis and/or the agent thattargets the prefrontal cortex) can also be an antidepressant, includingany of the agents in the SSRI (selective serotonin reuptake inhibitor)class.

Useful Chemical Compounds:

Agents useful in targeting the HPA axis include metyrapone andketoconazole. Metyrapone inhibits corticosterone synthesis by inhibitingthe 11β-hydroxylation step in the synthesis of adrenocorticosteroids(Sonino, In: Agarwal (Ed), Hormone antagonists, Walter de Gruyter,Berlin, pp 421-429, 1982; Haleem et al., Brain Res. 458, 339-347, 1988;Haynes, In: Gilman et al. (Eds), The Pharmacological Basis ofTherapeutics, eighth edition, Pergamon Press, New York, pp. 1431-1462,1990).

Metyrapone is commercially available and can be synthesized by contractmanufacturers (e.g., a pharmaceutical services company). In one scheme,metyrapone can be synthesized in a two-step process in which a startingmaterial is exposed to ultraviolet light (see, e.g., the syntheticpathway illustrated in FIG. 13).

The effect of metyrapone administration can be assessed by measuringplasma concentrations of cortitosterone. We investigated the effects ofthe corticosterone synthesis inhibitor metyrapone and ketoconazole oncocaine self-administration (see below). Pretreatment with metyraponeresulted in significant dose-related decreases in both plasmacorticosterone and ongoing cocaine self-administration, suggesting thatcorticosterone is involved in cocaine reward (see also Goeders et al.,Brain Res. 722:145-152, 1996).

Ketoconazole is an oral antimycotic agent with a broad spectrum ofactivity and low toxicity that is used in the treatment of fungaldisease (Sonino, In: Agarwal (Ed), Hormone Antagonists, Walter deGruyter, Berlin, pp 421-429, 1982; Thienpont et al., Experientia35:606-607, 1979). This drug also inhibits the 11β-hydroxylation and18-hydroxylation steps in the synthesis of adrenocorticosteroids(Engelhardt et al., Klin. Wochenschr. 63:607-612, 1985) and may alsofunction as a glucocorticoid receptor antagonist (Loose et al., J. Clin.Invest. 72:404-408, 1983). Furthermore, clinical trials have suggestedthat ketoconazole (as well as metyrapone) is effective in the treatmentof hypercortisolemic depression that is resistant to standardantidepressant therapy (Ghadirian et al., Biol. Psychiatry 37:369-375,1995; Murphy et al., J. Clin. Psychopharmacol. 11:121-126, 1991;Wolkowitz et al., Am. J. Psychiatry 150:810-812, 1993).

Agents that inhibit CRH include [Met18, Lys23, Glu27,29,40, Ala32,41,Leu33,36,38] CRF9-41, which is abbreviated as alpha-helical CRF(9-41)and has the sequenceAsp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Met-Leu-Glu-Met-Ala-Lys-Ala-Glu-Gln-Glu-Ala-Glu-Gln-Ala-Ala-Leu-Asn-Arg-Leu-Leu-Leu-Glu-Glu-Ala(SEQ ID NO:1)) and biologically active fragments or variants thereof(Rivier et al., Science 224:889, 1984). Another agent that inhibits CRHis [D-Phe12, Nle21,38, (αMeLeu37)] CRF(12-41), which is abbreviated asD-Phe CRF12-41, and biologically active fragments and variants thereof.Other agents that inhibit CRH include Astressin®; CP-154,526; NB127914,Antalarmin®; CRA1000; CRA1001, and Antisauvagine-30. See also U.S. Pat.Nos. 6,326,463; 6,323,312; and U.S. Pat. No. 4,594,329.

To inhibit ACTH, one can administer a sufficient amount of ACTH toinhibit ACTH through feedback inhibition or to down-regulate the ACTHreceptor. Compounds can be tested for their ability to affect ACTH invarious assays, including cell culture assays using, for example, ratanterior pituitary cells in monolayer culture (see Endocrinol. 91:562,1972).

Agents that inhibit activity within the HPA axis also include substanceP antagonists (e.g., [D-Arg1,D-Pro2,D-Trp7,9,Leu11]SP) and vasopressinantagonists.

As noted, in addition to metyrapone, ketoconazole, or another agent thatinhibits the HPA axis, the therapeutic agents of the present inventioncan include one or more agents that target the prefrontal cortex bytargeting GABA. Benzodiazepines (e.g., oxazepam) are one class of drugsuseful in that regard. Benzodiazepines are among the most widelyprescribed drugs for the pharmacological management of anxiety(Baldessarini, In: Hardman et al. (Eds), Goodman & Gilman's ThePharmacological Basis of Therapeutics, McGraw-Hill, New York, pp.399-430, 1996). As some of the major symptoms associated with cocainewithdrawal often include severe anxiety, restlessness and agitation(Crowley, In: Fisher et al. (Eds), Cocaine: Clinical and BiobehavioralAspects, Oxford University Press, New York, pp. 193-211, 1987; Gawin andEllinwood, Ann. Rev. Med. 40:149-161, 1989; Tarr and Macklin, PediatricClinics of North America 34:319-331, 1987), benzodiazepines may beuseful for alleviating these negative symptoms during the early stagesof withdrawal, and a benzodiazepine incorporated in the combinationtherapies described herein can be used to treat patients who exhibitthese and similar symptoms (i.e., anxiety, restlessness and agitation),whether in the context of an addiction or in connection with anotherevent (e.g., another neuropsychiatric event, menopause, or PMS). Thesedrugs are also useful in the emergency room for the treatment of some ofthe medical complications associated with cocaine intoxication sinceconvulsions are often apparent following an acute overdose. Theseseizures can be effectively treated with intravenous diazepam (Valium®)(Gay, J. Psychoactive Drugs 13:297-318, 1981; Tarr and Macklin,Pediatric Clinics of North America 34:319-331, 1987), and diazepam canbe used in the combination therapies described herein. Benzodiazepinereceptor expression can be assessed using methods known in the art. Forexample, receptors can be labeled with [³H]PK11195 (see Javaid et al.,Biol. Psychiatry 36:44-50, 1994; see also Chesley et al., J. Clin.Psychiatry 51:404-406, 1990). The data described below further suggeststhat benzodiazepines mediate certain aspects of cocaine reinforcement inrats.

Useful benzodiazepines or agents that target the prefrontal cortexinclude oxazepam, as noted above, as well as chlordiazepoxide,mirtazapine, atomoxetine, gabapentin (Neurontin™), muscimol, progabide,riluzole, baclofen, vigabatrin, valproic acid (Depakote™), tiagabine(Gabitril™), lamotrigine (Lamictal™), phenytoin (Dilantin™),carbamazepine (Tegretol™), and topiramate (Topamax™).

Other useful benzodiazepines include lorazepam (Ativan®), prazepam(Centrax®), flurazepam (Dalmane®), clonazepam (Klonopin®),chlordiazepoxide (Librium®), halazepam (Paxipam®), temezepam(Restoril®), clorazapate (Tranxene®), diazepam (Valium®), and alprazolam(Xanax®).

Where an agent that inhibits activity in the sympathetic nervous systemis included, so that agent can be a beta blocker or another type ofantihypertensive agent. More specifically, the agent can be sotalol(Betapace®), imolol (Blocadren®), carteolol (Cartrol®), carvedilol(Coreg®), nadolol (Corgard®), nadol/bendroflunetazide (Corzide®),propranolol (Inderal®), propranolol/HCTZ (Inderide®), betaxolol(Kerlone®), penbutolol (Levatol®), metoprolol (Lopressor®), labetalol(Normodyne®), acebutolol (Sectral®), atenolol/HCTZ (Tenoretic®),atenolol (Tenormin®), timolol/HCTZ (Timolide®), metoprolol (Toprol®),labetalol (Trandate®), pindolol (Visken®), bisoprolol (Zebeta®),bisoprolol/HCTZ (Ziac®), esmolol (Brevibloc®), or combinations thereof.

Alternatively, or in addition, where an agent that inhibits activity inthe sympathetic nervous system is included, it can be an SSRI. Currentlyavailable SSRIs, any of which or any combination of which can be used inthe present compositions and methods, include citalopram (Celexa®),escitalopram oxalate (Lexapro®), fluvoxamine (Luvox®), paroxetine(Paxil®), fluoxetine (Prozac®), and sertraline (Zoloft®).

Other useful agents that target the sympathetic nervous system, andwhich may be categorized as anxiolytic agents, are angiotensin IIinhibitors, and these agents include candasartan (Atacand®), eprosartan(Teveten®), irbesartan (Avapro®), losartan (Cozaar®), telmisartan(Micardis®), or valsartan (Diovan®).

Benzodiazepines are anxiolytic agents, and they may be incorporated inthe present compositions as either an agent that targets the prefrontalcortex and/or as an agent that inhibits the sympathetic nervous system.

The invention features pharmaceutically acceptable salts, solvates, orhydrates of any of the present compounds (i.e., of any of the compoundssuggested herein, generally or specifically, for use in combination),and prodrugs, metabolites, structural analogs, polymorphs, and otherpharmaceutically useful variants thereof, whether present as crystals,milled and stabilized as nanocrystals, or in a non-crystalline form.These other variants may be, for example, complexes containing thecompound (e.g., metyrapone) and a targeting moiety, as described furtherbelow, or a detectable marker (e.g., the compound may be joined to afluorescent compound or may incorporate a radioactive isotope). When inthe form of a prodrug, a compound may be modified in vivo (e.g.,intracellularly) after being administered to a patient or to a cell inculture. The modified compound (i.e., the processed prodrug) may beidentical to a compound described herein and will be biologically activeor have enough activity to be clinically beneficial. The same is true ofa metabolite; a given compound may be modified within a cell and yetretain sufficient biological activity to be clinically useful.

Nucleic Acid-Based Therapeutics:

The therapeutic agents useful in treating the conditions describedherein can also be nucleic acids. These nucleic acids can serve as thefirst agent that targets the HPA axis by inhibiting, directly orindirectly, the expression of CRH, ACTH, or cortisol, and they can serveas the second agent that targets the prefrontal cortex by increasingGABA. Where either or both of the first and second agents are used incombination with a third agent that inhibits the sympathetic nervoussystem, the “third” agent can be a nucleic acid that inhibits theexpression of a neurotransmitter or its cognate receptor within thesympathetic nervous system (e.g., the nucleic acid can inhibit theexpression of a β adrenergic receptor).

The nucleic acids can be “isolated” or “purified” (i.e., no longerassociated with some or all of the flanking nucleic acid sequences orcellular components with which the nucleic acid is naturally associatedin vivo). For example, with respect to a cell, tissue, or organism withwhich it was once naturally associated, a nucleic acid sequence usefulas a therapeutic agent can be at least 50% pure (e.g., 60%, 70%, 75%,80%, 85%, 90%, 95%, 98%, or 99% pure). Where a naturally occurring ormodified nucleic acid sequence (e.g., a cDNA) is administered, it mayinclude some of the 5′ or 3′ non-coding sequence associated with thenaturally occurring gene. For example, an isolated nucleic acid (DNA orRNA) can include some or all of the 5′ or 3′ non-coding sequence thatflanks the coding sequence (e.g., the DNA sequence that is transcribedinto, or the RNA sequence that gives rise to, the promoter or anenhancer in the mRNA). For example, an isolated nucleic acid can containless than about 5 kb (e.g., less than about 4 kb, 3 kb, 2 kb, 1 kb, 0.5kb, or 0.1 kb) of the 5′ and/or 3′ sequence that naturally flanks thenucleic acid molecule in a cell in which the nucleic acid naturallyoccurs. In the event the nucleic acid is RNA or mRNA, it is “isolated”or “purified” from a natural source (e.g., a tissue) or a cell culturewhen it is substantially free of the cellular components with which itnaturally associates in the cell and, if the cell was cultured, thecellular components and medium in which the cell was cultured (e.g.,when the RNA or mRNA is in a form that contains less than about 20%,10%, 5%, 1%, or less, of other cellular components or culture medium).When chemically synthesized, a nucleic acid (DNA or RNA) is “isolated”or “purified” when it is substantially free of the chemical precursorsor other chemicals used in its synthesis (e.g., when the nucleic acid isin a form that contains less than about 20%, 10%, 5%, 1%, or less, ofchemical precursors or other chemicals).

Nucleic acids useful in the compositions and methods described hereincan be double-stranded or single-stranded and can, therefore, either bea sense strand, an antisense strand, or a portion (i.e., a fragment) ofeither the sense or the antisense strand. The nucleic acids can besynthesized using standard nucleotides or nucleotide analogs orderivatives (e.g., inosine, phosphorothioate, or acridine substitutednucleotides), which can alter the nucleic acid's ability to pair withcomplementary sequences or to resist nucleases. The stability orsolubility of a nucleic acid can be altered (e.g., improved) bymodifying the nucleic acid's base moiety, sugar moiety, or phosphatebackbone. For example, the nucleic acids of the invention can bemodified as taught by Toulmé (Nature Biotech. 19:17, 2001) or Faria etal. (Nature Biotech. 19:40-44, 2001), and the deoxyribose phosphatebackbone of nucleic acids can be modified to generate peptide nucleicacids (PNAs; see Hyrup et al., Bioorganic & Medicinal Chemistry 4:5-23,1996).

PNAs are nucleic acid “mimics;” the molecule's natural backbone isreplaced by a pseudopeptide backbone and only the four nucleotide basesare retained. This allows specific hybridization to DNA and RNA underconditions of low ionic strength. PNAs can be synthesized using standardsolid phase peptide synthesis protocols as described, for example byHyrup et al. (supra) and Perry-O'Keefe et al. (Proc. Natl. Acad. Sci.USA 93:14670-675). PNAs of the nucleic acids described herein can beused in therapeutic and diagnostic applications. For example, PNAs canbe used as antisense or antigene agents for sequence-specific modulationof gene expression by, for example, inducing transcription ortranslation arrest or inhibiting replication.

The nucleic acids can be incorporated into a vector (e.g., anautonomously replicating plasmid or virus) prior to administration to apatient, and such vectors are within the scope of the present invention.The invention also encompasses genetic constructs (e.g., plasmids,cosmids, and other vectors that transport nucleic acids) that include anucleic acid of the invention in a sense or antisense orientation. Thenucleic acids can be operably linked to a regulatory sequence (e.g., apromoter, enhancer, or other expression control sequence, such as apolyadenylation signal) that facilitates expression of the nucleic acid.The vector can replicate autonomously or integrate into a host genome,and can be a viral vector, such as a replication defective retrovirus,an adenovirus, or an adeno-associated virus. In addition, when present,the regulatory sequence can direct constitutive or tissue-specificexpression of the nucleic acid.

The nucleic acids can be antisense oligonucleotides. While “antisense”to the coding strand of the targeted sequence, they need not bind to acoding sequence; they can also bind to a noncoding region (e.g., the 5′or 3′ untranslated region). For example, the antisense oligonucleotidecan be complementary to the region surrounding the translation startsite of an mRNA (e.g., between the −10 and +10 regions of a target geneof interest or in or around the polyadenylation signal). Moreover, geneexpression can be inhibited by targeting nucleotide sequencescomplementary to regulatory regions (e.g., promoters and/or enhancers)to form triple helical structures that prevent transcription of the genein target cells (see generally, Helene, Anticancer Drug Des. 6:569-84,1991; Helene, Ann. N.Y. Acad. Sci. 660:27-36, 1992; and Maher, Bioassays14:807-15, 1992). The sequences that can be targeted successfully inthis manner can be increased by creating a so-called “switchback”nucleic acid. Switchback molecules are synthesized in an alternating5′-3′,3′-5′ manner, such that they base pair with first one strand of aduplex and then the other, eliminating the necessity for a sizeablestretch of either purines or pyrimidines on one strand of a duplex.

Fragments having as few as 9-10 nucleotides (e.g., 12-14, 15-17, 18-20,21-23, or 24-27 nucleotides; siRNAs typically have 21 nucleotides) canbe useful and are within the scope of the invention.

In other embodiments, antisense nucleic acids can be anomeric nucleicacids, which form specific double-stranded hybrids with complementaryRNA in which, contrary to the usual b-units, the strands run parallel toeach other (Gaultier et al., Nucleic Acids Res. 15:6625-6641, 1987; seealso Tanaka et al., Nucl. Acids Res. 22:3069-3074, 1994). Alternatively,antisense nucleic acids can comprise a 2′-o-methylribonucleotide (Inoueet al., Nucleic Acids Res. 15:6131-6148, 1987) or a chimeric RNA-DNAanalogue (Inoue et al., FEBS Lett. 215:327-330, 1987).

Antibodies:

Antibodies and antigen binding fragments thereof useful as therapeuticagents in the present compositions. These antibodies may be of the Gclass (IgG), but IgM, IgD, IgA, and IgE antibodies can also be used;what is required is that the antibodies specifically bind a targetdescribed herein and alter that target—whether by enhancing orinhibiting its activity—in a way that, in accordance with our findings,confers a clinical benefit on a patient to whom they are administered.The antibodies can be polyclonal or monoclonal antibodies, and we usethe terms “antibody” and “antibodies” to refer to whole antibodies orfragments thereof that are, or that include, an antigen-binding domainof the whole antibody. For example, useful antibodies can lack the Fcportion; can be single chain antibodies; or can be fragments consistingof (or consisting essentially of) the variable, antigen-binding domainof the antibody. The antibodies can be humanized (by, for example, CDRgrafting) or fully human.

Methods of producing antibodies are well known in the art. For example,as noted above, human monoclonal antibodies can be generated intransgenic mice carrying the human immunoglobulin genes rather thanthose of the mouse. Splenocytes obtained from these mice (afterimmunization with an antigen of interest) can be used to producehybridomas that secrete human mAbs with specific affinities for epitopesfrom a human protein (see, e.g., WO 91/00906, WO 91/10741; WO 92/03918;WO 92/03917; Lonberg et al., Nature 368:856-859, 1994; Green et al.,Nature Genet. 7:13-21, 1994; Morrison et al. Proc. Natl. Acad. Sci. USA81:6851-6855, 1994; Bruggeman et al., Immunol. 7:33-40, 1993; Tuaillonet al., Proc. Natl. Acad. Sci. USA 90:3720-3724, 1993; and Bruggeman etal., Eur. J. Immunol 21:1323-1326, 1991).

The antibody can also be one in which the variable region, or a portionthereof (e.g., a CDR), is generated in a non-human organism (e.g., a rator mouse). Thus, the invention encompasses chimeric, CDR-grafted, andhumanized antibodies and antibodies that are generated in a non-humanorganism and then modified (in, e.g., the variable framework or constantregion) to decrease antigenicity in a human. Chimeric antibodies (i.e.,antibodies in which different portions are derived from different animalspecies (e.g., the variable region of a murine mAb and the constantregion of a human immunoglobulin) can be produced by recombinanttechniques known in the art. For example, a gene encoding the Fcconstant region of a murine (or other species) monoclonal antibodymolecule can be digested with restriction enzymes to remove the regionencoding the murine Fc, and the equivalent portion of a gene encoding ahuman Fe constant region can be substituted therefor (see EuropeanPatent Application Nos. 125,023; 184,187; 171,496; and 173,494; see alsoWO 86/01533; U.S. Pat. No. 4,816,567; Better et al., Science240:1041-1043, 1988; Liu et al., Proc. Natl. Acad. Sci. USA84:3439-3443, 1987; Liu et al., J. Immunol. 139:3521-3526, 1987; Sun etal., Proc. Natl. Acad. Sci. USA 84:214-218, 1987; Nishimura et al.,Cancer Res. 47:999-1005, 1987; Wood et al., Nature 314:446-449, 1985;Shaw et al., J. Natl. Cancer Inst. 80:1553-1559, 1988; Morrison et al.,Proc. Natl. Acad. Sci. USA 81:6851, 1984; Neuberger et al., Nature312:604, 1984; and Takeda et al., Nature 314:452, 1984).

An antigen-binding fragment of the invention can be: (i) a Fab fragment(i.e., a monovalent fragment consisting of the VL, VH, CL and CH1domains); (ii) a F(ab′)₂ fragment (i.e., a bivalent fragment containingtwo Fab fragments linked by a disulfide bond at the hinge region); (iii)a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragmentconsisting of the VL and VH domains of a single arm of an antibody, (v)a dAb fragment (Ward et al., Nature 341:544-546, 1989), which consistsof a VH domain; and (vi) an isolated complementarity determining region(CDR).

Expression vectors can be used to produce the proteins of the invention,including antibodies, ex vivo (e.g., the proteins of the invention canbe purified from expression systems such as those described herein) orin vivo (in, for example, whole organisms).

Formulations and Dosages:

The identified agents that target the HPA axis, the prefrontal cortexand/or the sympathetic nervous system can be administered to a patientat therapeutically effective doses to prevent, treat or ameliorate anyof the disorders or conditions described herein (e.g., an addiction,obesity, post-traumatic stress disorder or an associated condition). Atherapeutically effective dose refers to an amount of the agent orcombination of agents sufficient to improve at least one of the signs orsymptoms of the disorder or condition.

Many of the agents useful in the context of the present invention havebeen used previously to treat patients for other reasons. Where dosinginformation is available, it can be used to help determine effectivedoses of the agents in the presently described combinations. The doseused to treat a patient for an addiction, one of the other disordersdescribed herein, and/or a related condition, can be the same as thedose that has been used previously for another indication. The doses mayalso differ. For example, the effective dosages required in connectionwith the combination therapies described herein may be less than thosepreviously proven safe and effective.

Toxicity and therapeutic efficacy of the agents described herein can bedetermined, as necessary, by standard pharmaceutical procedures in cellcultures or experimental animals. For example, laboratory animals suchas rodents and non-human primates can be used to determine the LD₅₀ (thedose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index, whichcan be expressed as the ratio LD₅₀:ED₅₀. Compounds that exhibit largetherapeutic indices are typically preferred.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays (e.g., assays designedto determine whether a nucleic acid, nucleic acid-based agent, or aprotein such as an antibody inhibits (or stimulates) the expression oractivity of the ligand or receptor it is intended to inhibit (orstimulate)).

A dose can be formulated in animal models to achieve a circulatingplasma concentration range that includes the IC₅₀ (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses (e.g.,therapeutically effective doses) in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.

One of the greatest concerns in the treatment of drug addiction is thehigh rate of recidivism. This phenomenon can be tested in animals duringreinstatement, which is a widely regarded preclinical model of thepropensity to relapse to drug taking, and animal models of reinstatementcan be used to further determine and define effective doses of theagents described herein. For example, animals can be taught toself-administer a drug until stable drug intake is maintained and thensubjected to prolonged periods of extinction training or abstinence.Once the criteria for extinction are met, or following a specifiedperiod of abstinence, the ability of specific stimuli to reinstateresponding on the manipulandum previously associated with the deliveryof drug infusions is taken as a measure of drug seeking. Thisreinstatement of drug-seeking behavior can be elicited by priminginjections of the drug itself in rats and monkeys (Stewart, J.Psychiatr. Neurosci. 25:125-136, 2000) or by exposure to brief periodsof intermittent electric footshock in rats (Shaham et al., Brain Res.Rev. 33:13-33, 2000; Stewart, J. Psychiatr. Neurosci. 25:125-136, 2000).Acute re-exposure to the self-administered drug (de Wit, Exp. Clin.Psychopharmacol. 4:5-10, 1996) and exposure to stress (Shiffman andWills, Coping and Substance Abuse, Academic Press, Orlando, 1985; Lamonand Alonzo, Addict. Behav. 22:195-205, 1997; Brady and Sonne, Alc. Res.Health 23:263-271, 1999; Sinha, Psychopharmacol. 158:343-359, 2001; andSinha et al., Psychopharmacol. 142:343-351, 1999), or simply thepresentation of stress-related imagery (Sinha et al., Psychopharmacol.158:343-359, 2000), have also been identified as potent events forprovoking relapse to drug seeking in humans.

In the studies described below, we initially found a dose of each ofmetyrapone and oxazepam that reduced cocaine self-administration withoutproducing nonspecific debilitating effects on other behaviors. We thenreduced the dose by one-half until we found a dose of each drug that nolonger affected cocaine self-administration or any other observablebehaviors (i.e., an ineffective dose). When we then combined theineffective doses of the two drugs, cocaine self-administration wasreduced. This suggests that although the two drugs produce their effectsthrough different mechanisms, the effects are additive. Thus, weconcluded that combining drugs that affect the HPA axis throughdifferent mechanisms can produce an additive effect on cocaine reward.Furthermore, by combining these drugs at concentrations that have noeffect when the drugs are administered alone, we can minimize thepotential toxic side effects (e.g., excessive decreases in plasmacortisol with metyrapone and the abuse liability of benzodiazepines)that may be associated with these compounds. Accordingly, thecompositions of the present invention may include combinations oftherapeutic agents, one or both of which are present at a dosage levellower than that which would be required to achieve an effect had theagent been administered alone; the dosages may be additive.

Pharmaceutical compositions for use in accordance with the presentinvention can be formulated in any conventional manner using one or morephysiologically acceptable carriers or excipients. Thus, the agents,including compounds and their physiologically acceptable salts andsolvates, can be formulated for administration by or oral or parenteraladministration.

For oral administration, the pharmaceutical compositions can take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets can be coated by methods well known in the art. Liquidpreparations for oral administration can take the form of, for example,solutions, syrups or suspensions, or they can be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations can be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations can also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration can be suitably formulated to givecontrolled release of the active compound(s) (which we may refer toherein as “therapeutic agent(s)”). The agents, including compounds(e.g., small organic molecules) can be formulated for parenteraladministration by injection (e.g., by bolus injection or continuousinfusion). Formulations for injection can be presented in unit dosageform, (e.g., in ampoules or in multi-dose containers) with an addedpreservative. The compositions can take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and can containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient can be in powder form forconstitution with a suitable vehicle (e.g., sterile pyrogen-free water)before use.

In addition to the formulations described previously; the agents canalso be formulated as a depot preparation. Such long acting formulationscan be administered by implantation (for example, subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, theagents can be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

The compositions can also be formulated for other routes ofadministration, including intranasal, topical, and mucosal (e.g., bysublingual administration).

The compositions can, if desired, be presented in a pack or dispenserdevice which can contain one or more unit dosage forms containing theactive ingredient. The pack can for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device can beaccompanied by instructions for administration. Various presentationforms (e.g., presentation by way of packs and dispensers) are within thescope of the present invention.

Nucleic acids, including antisense nucleic acids, can also beadministered systemically and, if so, may be modified to target selectedcells within the HPA axis, the prefrontal cortex and/or the sympatheticnervous system. For example, antisense nucleic acids can be linked toantibodies or other proteins (e.g., receptor ligands) that willspecifically bind to cell surface receptors or other componentsassociated with the target cell type. Similarly, the nucleic acids caninclude agents that facilitate their transport across the cell membrane(see, e.g., Letsinger et al., Proc. Natl. Acad. Sci. USA 86:6553-6556,1989; Lemaitre et al., Proc. Natl. Acad. Sci. USA 84:648-652, 1987; andWO 88/09810) or the blood-brain barrier (see, e.g., WO 89/10134). Inaddition, nucleic acids can be modified with intercalating agents (Zon,Pharm. Res. 5:539-549, 1988). Antisense nucleic acids can also bedelivered to cells using the vectors described herein. To achievesufficient intracellular concentrations of antisense nucleic acids, onecan express them in vectors having a strong promoter (e.g., a strong polII or pol III promoter).

In specific embodiments, the invention features pharmaceuticalcompositions that include a first agent that targets the HPA axis and asecond agent that targets the prefrontal cortex. The first agent can bean agent that inhibits CRH, that inhibits ACTH, and/or that inhibitscortisol and the second agent can be an agent that increases theexpression, secretion, or activity of GABA, is a GABA mimic, and/orinhibits GABA metabolism. Either the first and/or the second agent canbe a chemical compound. For example, the first agent can be metyrapone(Metopirone®) or ketoconazole (Nizoral®) or a salt, solvate, hydrate,prodrug, structural analog, or polymorph thereof. The second agent canbe a benzodiazepine (e.g., oxazepam or chlordiazepoxide) or a salt,solvate, hydrate, prodrug, structural analog, or polymorph thereof. Thesecond agent can also be mirtazapine or atomoxetine or salts, solvates,hydrates, prodrugs, structural analogs, or polymorphs thereof. Anotheruseful second agent is gabapentin (Neurontin™) or a salt, solvate,hydrate, prodrug, structural analog, or polymorph thereof, or ismuscimol or baclofen or salts, solvates, hydrates, prodrugs, structuralanalogs, or polymorphs thereof. Additional useful second agents are:progabide, riluzole, baclofen, vigabatrin, valproic acid (Depakote™),tiagabine (Gabitril™), lamotrigine (Lamictal™), phenytoin (Dilantin™),carbamazepine (Tegretol™), and topiramate (Topamax™) or salts, solvates,hydrates, prodrugs, structural analogs, or polymorphs thereof. Any ofthe pharmaceutical compositions can be formulated for oraladministration or for intravenous administration. The amount of thefirst agent or the amount of the second agent in a unit dosage can beless than the amount of the first agent or the second agent currently ortypically prescribed for a patient requiring the same unit dosage.Combining the agents may allow them to be administered at dosages thatare lower than expected given current, commonly prescribed dosages. Forexample, a pharmaceutical composition can include about 5-60 mg ofoxazepam and about 250-1000 mg of metyrapone (Metopirone®) in unitdosage form. Any of these compositions can further include a third agentthat inhibits activity in the sympathetic nervous system. The thirdagent can be a beta blocker (e.g., sotalol (Betapace®), imolol(Blocadren®), carteolol (Cartrol®), carvedilol (Coreg®), nadolol(Corgard®), nadol/bendroflunetazide (Corzide®), propranolol (Inderal®),propranolol/HCTZ (Inderide®), betaxolol (Kerlone®), penbutolol(Levatol®), metoprolol (Lopressor®), labetalol (Normodyne®), acebutolol(Sectral®), atenolol/HCTZ (Tenoretic®), atenolol (Tenormin®),timolol/HCTZ (Timolide®), metoprolol (Toprol®), labetalol (Trandate®),pindolol (Visken®), bisoprolol (Zebeta®), bisoprolol/HCTZ (Ziac®), oresmolol (Brevibloc®)) or other anxiolytic compound (e.g., an SSRI suchas citalopram (Celexa®), escitalopram oxalate (Lexapro®), fluvoxamine(Luvox®), paroxetine (Paxil®), fluoxetine (Prozac®), or sertraline(Zoloft®)). The anxiolytic compound or agent can also be an angiotensinII inhibitor (e.g., candasartan (Atacand®), eprosartan (Teveten®),irbesartan (Avapro®), losartan (Cozaar®), telmisartan (Micardis®), orvalsartan (Diovan®)).

Concentrated compositions, suitable for shipment, storage, and laterdilution are also within the invention.

The pharmaceutical compositions described above can be used in themethods described herein, including those that follow, and for thepurposes of use described below (e.g. for use in the preparation of amedicament and/or in the preparation of a medicament for treating adisorder or condition described herein).

Methods of Treatment:

As noted, the compositions described herein can be used to treatpatients suffering from a disorder associated with aberrant activity inthe HPA axis. The treatment methods can include various steps, one ofwhich can constitute identifying a patient in need of treatment.Physicians are well able to examine and diagnose patients suspected ofsuffering from addiction and/or another of the conditions describedherein. Following a diagnosis, which may be made in the alternative, thephysician can prescribe a therapeutically effective amount of acomposition (e.g., a pharmaceutical composition comprising a first agentthat targets the HPA axis and a second agent that targets the prefrontalcortex). The patient may have, or be diagnosed as having, an addictionto a substance such as alcohol, a chemical stimulant, a prescription (orprescribed) pain reliever, or a naturally-occurring plant-derived drug.The chemical stimulant can be cocaine, an amphetamine, methamphetamine,or crystalline methylamphetamine hydrochloride, or methylphenidate.Where analogs of specific drugs are addictive, addictions to thoseanalogs can also be treated.

The drug can also be a barbiturate (e.g., thiamyl (Surital®), thiopental(Pentothal®), amobarbital (Amyta®), pentobarbital (Nembutal®),secobarbital (Seconal®), Tuinal (an amobarbital/secobarbital combinationproduct), butalbital (Fiorina®), butabarbital (Butisol®), talbutal(Lotusate®), aprobarbital (Alurate®), phenobarbital (Luminal®), andmephobarbital (Mebaral®)), or opiate (e.g., heroin, codeine,hydrocodone).

Naturally-occurring plant-derived drugs include marijuana and tobacco.The compositions described herein can be used to treat patients addictedto these substances generally and/or to a more specific ingredienttherein (e.g., the nicotine in tobacco). The addiction may also manifestas addiction to an activity such as gambling, sex or a sexual activity,or overeating (which may be associated with an eating disorder or mayresult in obesity). More generally, eating and sleeping disorders areamong those amenable to treatment with the present compositions. Eatingdisorders include anorexia nervosa, bulimia nervosa, binge eatingdisorder and eating disorders not otherwise specified (EDNOS). Severalstudies have examined the function of the HPA axis in anorexia nervosa.A principal finding is that of hypercortisolism, associated withincreased central corticotrophin-releasing hormone levels and normalcirculating levels of adrenocorticotropic hormone. While anorexianervosa can be difficult to diagnose, patients with this disorderpresent with endocrine dysfunction, often evident as amenorrhea,abnormal temperature regulation, abnormal growth hormone levels, andabnormal eating. The present methods can include a step of identifying apatient in need of treatment, and these characteristics would be, orwould likely be among, those used by physicians to diagnose anorexianervosa.

The present compositions can be used to treat patients who have PraderWilli syndrome, and methods of treating such patients are within thescope of the invention. Sleep disorders include insomnia, sleep apneasleep disorder, Restless Legs Syndrome (RLS) and Periodic Limb MovementDisorder (PLMD), and narcolepsy.

Other patients amenable to treatment include those suffering fromanxiety (which may be associated with panic disorder, obsessivecompulsive disorder (OCD), post-traumatic stress disorder (PTSD), socialanxiety disorder, or may be a generalized anxiety disorder). Where thecondition is depression, it may be depression associated with majordepressive disorder or dysthymia, bipolar depression, or may beassociated with a medical condition or substance abuse. The risk ofdeveloping depression or other major affective disorders is determinedby a complex interplay between genetic susceptibility, environmentalexposures, and aging.

Other patients amenable to treatment include those suffering fromschizophrenia; those with an attention-deficit disorder (e.g., ADD orADHD); those experiencing menopause; and those suffering from amenstrual cycle-related syndrome (e.g., PMS).

The disorders and events described herein may be variously categorizedand may be related to one another in various ways. For example, socialanxiety may contribute to an eating disorder and otheranxiety-associated conditions, such as PTDSs, may manifest as a sleepdisorder. Patients diagnosed as clinically depressed may also experiencesleep disorders. Addiction, which has been characterized as aprogressive disorder, may begin with the self-administration of aprescription or non-prescription drug to alleviate a symptom of anotherneuropsychiatric disorder. For example, a patient may self-administeralcohol or marijuana in the event of a depression or anxiety or asleep-aid to treat the difficulty in sleeping as a result thereof. Therelationships between the disorders and related conditions or symptomsmay flow in different directions as well. For example, chronicactivation of the HPA axis in insomnia puts insomniacs at risk not onlyfor mental disorders (i.e., chronic anxiety and depression), but alsofor significant medical morbidity associated with such activation.Insomnia is, by far, the most commonly encountered sleep disorder inmedical practice. Either as a symptom of various psychiatric or medicaldisorders or as the result of a stressful situation, chronic and severeinsomnia is perceived by the patient as a distinct disorder (seeVgontzas et al., J. Clin. Endocrinol. Metabl. 86:3787-3794, 2006). Sleepdisorders, including insomnia, can occur during menopause or when apatient is suffering from PMS.

Just as there can be some overlap in the categorization of theindications described herein, there can be some overlap in the nature ofthe agents applied and/or the manner in which they are categorized. Forexample, and as noted above, benzodiazepines can be used as the “second”agent to target the prefrontal cortex. Benzodiazepines can also becategorized as anti-anxiety drugs and therefore are suitable as the“third” agent described herein.

The success of the treatment can be assessed in a variety of ways,including objective measures (e.g., where the patient is addicted to asubstance or activity, a reduction in the frequency or severity of drugself-administration or other addictive activity), a general improvementin health (e.g., an improvement in blood pressure, kidney function,liver function, or blood count) and/or subjective measures (e.g., apatient's report of reduced craving for a substance or activity or abetter sense of well-being (e.g., where the patient suffers from anxietyor an anxiety-related disorder, a report of reduced anxiety, an improvedmood, a greater sense of well-being, or an improved ability to cope withdaily stressors)). Where the condition treated is an eating disorder orsleep disorder, treatment can be assessed by judging the effectivereturn of (or return toward) normal eating or sleeping patterns.

In specific embodiments, the invention features methods of treating apatient who is suffering from a disorder associated with aberrantactivity in the HPA axis. The method can include the steps of: (a)identifying a patient in need of treatment; and (b) administering to thepatient a therapeutically effective amount of a composition describedherein. The disorder can include addiction, anxiety, schizophrenia, ordepression; the disorder can be an addiction to a substance (e.g., achemical stimulant such as an opiate (e.g., heroin, codeine,hydrocodone, or analogs thereof), nicotine, alcohol, prescription painreliever, or naturally-occurring plant-derived drug, such as nicotine).The chemical stimulant can also be cocaine, an amphetamine, amethamphetamine, methylphenidate, or analogs thereof.

The disorder can also be an addiction to an activity such as gambling orengaging in a sexual activity or excessive eating.

Where the patient is suffering from anxiety, the anxiety may beassociated with a panic disorder, an obsessive compulsive disorder(OCD), a post-traumatic stress disorder (PTSD), a social anxietydisorder, or a generalized anxiety disorder. Where the patient issuffering from depression, the depression can be associated with majordepressive disorder or dysthymia, with a bipolar depression, or amedical condition or substance abuse. As noted, the disorder can also bean eating disorder or a sleep disorder or a disruptive behaviordisorder.

The methods can be carried out in treating a patient who is sufferingfrom an unwanted symptom of menopause or the menstrual cycle by: (a)identifying a patient in need of treatment; and (b) administering to thepatient a therapeutically effective amount of a composition describedherein. The amounts of the compositions delivered are therapeuticallyeffective, with effectiveness judged by relief in symptoms, which mayinclude anxiety, depression, or difficulty sleeping.

The invention features the use of the compositions described herein inthe preparation of a medicament. The invention further features the useof the compositions described herein in the preparation of a medicamentfor the treatment of obesity; an eating disorder; a sleep disorder;depression; a disruptive behavior disorder; schizophrenia; and/oranxiety, regardless of context.

EXAMPLES Effects of Low Dose Combination Pharmacotherapy on CocaineSelf-Administration in Rats

The studies described here examine a combination pharmacotherapy,consistent with that described herein, for the treatment of addiction(more specifically, cocaine abuse). Using this approach, two compounds,which are believed to use divergent mechanisms of action to ultimatelyproduce similar effects on the body's responses to stressors, areadministered together at doses that are ineffective, or much lesseffective, alone. Adult male Wistar rats were trained under a multiple,alternating schedule of cocaine and food self-administration. Thisschedule consisted of alternating periods of cocaine access and foodreinforcement. In some instances, as described further below, threedoses of cocaine (0.125, 0.25, or 0.50 mg/kg/infusion) were tested. Ratswere also periodically trained with saline substitution (cocaineextinction) and food extinction during the same session.

These studies support the conclusion that pretreatment with thecorticosterone synthesis inhibitors metyrapone and ketoconazole, thebenzodiazepines chlordiazepoxide, alprazolam and oxazepam, and the CRHreceptor antagonist CP-154,526 all decrease cocaine self-administrationand the reinstatement of extinguished cocaine seeking in rats. Webelieve the combination pharmacotherapy reduces the likelihood ofrelapse by attenuating cue-induced increases in activity within the HPAaxis, thereby reducing the cue-induced secretion of CRH, ACTH andcortisol (corticosterone), and by decreasing cue-induced alterations inactivity in the prefrontal cortex.

Combinations Tested

The combinations of drugs we tested include:

(1) metyrapone and oxazepam; (2) ketoconazole and alprazolam; (3)ketoconazole and oxazepam; (4) metyrapone and alprazolam; (5) muscimoland CP-154,526; and (6) muscimol and metyrapone. The drug combinationsconsist of at least one drug from each class (e.g., metyrapone andoxazepam). As noted, the drugs were combined at doses below theirnormally effective doses, and an additive or synergistic effect emerged.

Training to Self-Administer Cocaine

In our model, rats were exposed to alternating 15-minute periods ofaccess to cocaine self-administration and food reinforcement. Food wasused to control for potential nonspecific, ataxis effects of the drugsand combinations. The ideal drug or drug combination is one that reducescocaine self-administration without affecting food-maintainedresponding. The other preclinical model we used is the cue-inducedreinstatement of extinguished cocaine seeking model of relapse. In thismodel, rats are trained to self-administer cocaine and the ability ofconditioned cues in the environment to reinstate extinguished respondingis assessed and taken as a measure of relapse.

More specifically, adult male Wistar rats were implanted with chronicjugular catheters. Following recovery from surgery, the rats weretrained to respond under a multiple, alternating schedule of foodreinforcement and cocaine self-administration. Food-maintainedresponding was used to control for the non-specific motor effects of thevarious treatments. During the food component of the schedule, thestimulus light located above the food response lever was illuminated toindicate the availability of food reinforcement. Initially, eachdepression of the food response lever resulted in a brief darkening ofthe food stimulus light (0.6 seconds) and the delivery of a food pellet(45 mg). A 25-second timeout followed the delivery of each food pellet.During this timeout, the stimulus light was darkened and responses onthe food lever were counted but had no scheduled consequences.Responding on the other (cocaine) lever during the food component alsohad no scheduled consequences. The response requirement for the foodlever was gradually increased over several sessions from continuousreinforcement to a fixed-ratio four schedule whereby four responses wererequired for food presentation. Following 15 minutes of access to food,all stimulus lights in the chamber were darkened for a 1-minute timeout.Following the timeout, the stimulus light above the cocaine responselever was illuminated to indicate the availability of cocaine (0.125,0.25, or 0.5 mg/kg/infusion). Initially, each depression of the cocaineresponse lever resulted in a brief darkening of the stimulus light andan infusion of cocaine (200 μl delivered over 5.6 seconds). A 20-secondtimeout period followed each infusion. The response requirement forcocaine was gradually increased to a fixed-ratio four schedule ofreinforcement. After 15 minutes of access to cocaine and a 1-minutetimeout, the rats were again allowed 15 minutes access to the foodcomponent of the schedule. Access to food and cocaine alternated in thismanner every 15 minutes during the two hour behavioral sessions so thateach rat was exposed to food and cocaine for four 15-minute periodseach. Each behavioral session began with 15 minutes access to eitherfood or cocaine, and this alternated daily. Stable baselines ofresponding were established when the total number of cocaine and foodpresentations, as well as the number of presentations during each of thefour exposures each session, varied less than 10% for three consecutivesessions. At least three different doses of cocaine (e.g., 0.125, 0.25,and 0.5 mg/kg/infusion) were tested. Rats were first trained toself-administer 0.25 mg/kg/infusion, our standard dose of cocaine. Whenresponding stabilized, the dose was changed to 0.125 or 0.5mg/kg/infusion as appropriate. We have found that initially trainingrats with this moderated dose of cocaine (i.e., 0.25 mg/kg/infusion)hastens stability with the lower dose (i.e., 0.125 mg/kg/infusion).

Once stable baselines of responding were obtained, dose-response curvesfor the various compounds were individually generated for each rat. Ratswere treated with each dose at least twice with a minimum of two days ofbaseline cocaine self-administration interspersed between each test.Each group of rats was tested with only two of the test compounds tominimize potential carryover effects. The minimally effective dose thatreduced cocaine self-administration by at least 50% without affectingfood-maintained responding (i.e., the high dose) was determined for eachcompound. The dose selected for the drug combination experiments wasone-half of the minimally effective dose, and this dose had to alsoproduce less than a 10% decrease in cocaine self-administration (i.e.,an ineffective dose). If one-half of the minimally effective dosereduced cocaine self-administration by more than 10%, then the dose wasonce again reduced by one-half. For example, the minimally effectivedose of ketoconazole was 25 mg/kg, and we have successfully used 12.5mg/kg in our studies with alprazolam and oxazepam. This dose (12.5mg/kg) has no effect on cocaine- or food-maintained responding whentested alone, but significantly reduces cocaine self-administration whencombined with a similarly ineffective dose of alprazolam (i.e., 1.0mg/kg, ip) or oxazepam (10 mg/kg, ip). This rationale guided theselection of the doses of each of the compounds in the combinationstudies. Each experimental group consisted of between 8 and 10 rats.

Cue-Induced Reinstatement of Extinguished Cocaine Seeking

The experiments described herein were designed to investigate whether ornot drug combinations identified as effective in reducing cocaineself-administration would also block the ability of conditioned cues toreinstate extinguished cocaine-seeking behavior. Adult male Wistar ratswere implanted with chronic jugular catheters and trained toself-administer cocaine (0.25 mg/kg/infusion) by pressing one of theresponse levers in the experimental chamber (i.e., the “active” or“cocaine” lever) under a fixed-ratio four (FR4) schedule ofreinforcement during daily 2-hour sessions conducted 5 days per week. Atthe start of each session, both levers were extended into the chamberand the stimulus light above the active lever was illuminated toindicate the availability of cocaine. Initially, each depression of theactive lever resulted in an intravenous infusion of cocaine and theconcurrent presentation of a house light and tone compound stimulus(i.e., the conditioned cue or secondary reinforcer). A 20-second timeoutperiod followed each infusion. The stimulus light above the active leverand the house light and tone compound stimulus were extinguished duringthe timeout period, and the light above the active lever was illuminatedonce the timeout ended. When responding on the active lever varied lessthan 20% for two consecutive days, the response requirement wasincreased to FR2. When similar stability was observed under the FR2schedule or reinforcement, the response requirement was increased to thefinal ratio of four. The criteria for stable responding under the FR4schedule of reinforcement was a minimum of 10 days of exposure to thisschedule that concluded with at least three consecutive days whenresponding varied by less than 10%. Responses on the inactive lever werecounted but resulted in no programmed consequences at any time. Oncestable cocaine self-administration was observed, rats were exposed toextinction; the rats were placed into the behavioral chambers, butresponding on the “cocaine” (active) lever produced no programmedconsequences. Extinction training continued until responding decreasedto less than 20% of baseline self-administration. Then reinstatementtesting commended. The rats were placed into the experimental chambers,both response levers were extended into the chamber, and the stimuluslight above the “active” lever was illuminated as duringself-administration training. During reinstatement, responding on the“active” lever resulted in a 5.6-second presentation of the conditionedreinforcer (i.e., the house light and tone compound stimulus that hadbeen paired with cocaine during self-administration). Responses on the“inactive” lever were counted but resulted in no scheduled consequences.Responding on the “active” lever during reinstatement testing was takenas an index of cocaine-seeking behavior. Each experimental groupconsisted of 8 to 10 rats.

The Effect of Metyrapone and Oxazepam on Intravenous Self-Administrationof Cocaine

These experiments were designed to determine the effects of acombination of metyrapone and oxazepam on intravenous cocaineself-administration in rats responding under a multiple, alternatingschedule of food reinforcement and cocaine self-administration. Theresults are depicted in the graph of FIG. 1A. The first bar to the left(“Ext”) shows the results of extinction when responding on the “active”lever only resulted in infusions of saline. The second bar (“Veh”)depicts the number of cocaine infusions self-administered followingpretreatment with the vehicle (5% emulphor in 0.9% saline) for thetreatment drugs. The “Met-high” bar shows the number of infusions ofcocaine following pretreatment with the high dose of metyrapone (25-175mg/kg, ip), while the “OX-high” bar depicts the number of cocaineinfusions self-administered following pretreatment with the high dose ofoxazepam (5-80 mg/kg, ip).

Both metyrapone and oxazepam reduced cocaine self-administration withoutaffecting food-maintained responding at these doses. The “Met-low” and“OX-low” bars represent responding following pretreatment with the low,ineffective doses (oxazepam 5-25 mg/kg, ip; metyrapone 25-50 mg/kg, ip)of metyrapone and oxazepam alone. Clearly, these doses did notsignificantly affect cocaine self-administration (or food-maintainedresponding) when administered alone. The “COM-low” bar depicts thenumber of cocaine infusions self-administered following the delivery ofthe combination pharmacotherapy (i.e., an injection consisting of theineffective doses of metyrapone and oxazepam). As can be seen, thecombination pharmacotherapy consisting of metyrapone and oxazepamreduced cocaine self-administration to levels seen when only saline wasdelivered when the active lever was pressed during extinction. Thecombination pharmacotherapy reduced cocaine self-administration toextinction levels without affecting food-maintained responding,suggesting that the combination was reducing the motivation to seekcocaine without affecting responding or the motivation for anotherreinforcer (i.e., food).

FIG. 1B depicts the same data as shown in FIG. 1A, but the data arepresented as the percentage of baseline infusions under the conditionstested. The “high” dose of metyrapone and oxazepam reduced cocaineself-administration to less than 50% of baseline self-administration,while the “low” doses only reduced self-administration by 10% or less.As in FIG. 1A, the combination of the low doses of oxazepam andmetyrapone reduced cocaine self-administration to levels seen duringextinction.

FIG. 2A depicts experiments designed to investigate the effects of thecombination of the ineffective doses of metyrapone and oxazepam oncocaine self-administration when different groups of rats were trainedto self-administer different doses of cocaine. It is important todetermine whether or not the rats could overcome the effects of thecombination when higher doses of cocaine were available. This would beanalogous to a cocaine addict increasing his or her intake of cocaine toovercome the effects of the combination pharmacotherapy. The numbers onthe X-axis represent the three doses of cocaine that wereself-administered. “Saline” shows the number of infusionsself-administered when only saline was in the syringe (i.e.,extinction). “Vehicle” represents the number of cocaine infusionsself-administered when the vehicle (5% emulphor in 0.9% saline) for thetreatment of drugs was delivered prior to the start of the cocaineself-administration session. “Combo” depicts the number of cocaineinfusions self-administered following pretreatment with the combinationof the ineffective doses of metyrapone and oxazepam. Clearly, thiscombination reduced cocaine self-administration to extinction levelsregardless of the dose of cocaine that was available forself-administration. This indicates that the effects of the combinationpharmacotherapy would not easily be overcome by increasing the intake ordose of cocaine.

FIG. 2B depicts the same data as in FIG. 2A, but the data are presentedas the percentage of baseline infusions under the different conditions.FIG. 2A shows that the combination of the low doses of oxazepam andmetyrapone reduced cocaine self-administration to levels seen duringextinction regardless of the dose of cocaine that was available forself-administration.

We also conducted experiments to determine the effect of a combinationof metyrapone and oxazepam on the cue-induced reinstatement ofextinguished cocaine seeking in rats. We used an animal model of therelapse to cocaine seeking. Referring to FIG. 10, the bar labeled “SA”depicts the number of responses made on the “active” lever duringcocaine self-administration. The bar labeled “EXT” depicts the number ofresponses on the “active” lever during extinction when responding onthis lever only resulted in infusions of saline. The third bar, “VEH”,represents responding on the “active” lever during reinstatement testingfollowing pretreatment with the vehicle (5% emulphor in 0.9% saline) forthe treatment drugs. The last bar, “COMBO”, depicts the number ofresponses on the “active” lever during reinstatement testing followingthe delivery of the combination pharmacotherapy (i.e., an injectionconsisting of the ineffective doses of metyrapone and oxazepain asdetermined in the cocaine self-administration experiments (see FIG. 1A).The combination pharmacotherapy reduced cocaine seeking (i.e.,responding on the active lever during reinstatement) to levels seen whenonly saline was delivered when the active lever was pressed duringextinction. The combination therapy reduced reinstatement (relapse) toextinction levels without affecting food-maintained responding. Thissuggests that the combination reduced the motivation to seek cocainewithout affecting responding or motivation for another reinforcer (i.e.,food).

The Effect of Ketoconazole and Alprazolam on IntravenousSelf-Administration of Cocaine

These experiments were designed to determine the effects of acombination of ketoconazole and alprazolam on intravenous cocaineself-administration in rats responding under a multiple, alternatingschedule of food reinforcement and cocaine self-administration. The dataare presented in FIG. 3. The solid bar (“VEH”) depicts the number ofcocaine infusions self-administered following pretreatment with thevehicle (5% emulphor in 0.9% saline). The open bar (“EXT”) shows theresults of extinction when responding on the “active” lever onlyresulted in infusions of saline. The “ALP” (striped) and “KETO” (lightlyshaded) bars represent self-administration following pretreatment withthe low, ineffective doses (alprazolam 0.2-2 mg/kg, ip; ketoconazole5-75 mg/kg, ip) of alprazolam and ketoconazole alone. Clearly, thesedoses did not significantly affect cocaine self-administration (orfood-maintained responding) when administered alone. The “COMBO” (smallstriped) bar depicts the number of cocaine infusions self-administeredfollowing the delivery of the combination pharmacotherapy (i.e., aninjection consisting of the ineffective doses of alprazolam andketoconazole). As can be clearly seen, the combination pharmacotherapyconsisting of alprazolam and ketoconazole reduced cocaineself-administration to levels seen when only saline was delivered whenthe active lever was pressed during extinction. The combinationpharmacotherapy reduced cocaine self-administration to extinction levelswithout affecting food-maintained responding. This suggests that thecombination was reducing the motivation to seek cocaine withoutaffecting responding or motivation for another reinforcer (i.e., food).These data also demonstrate that the effects of the combinationpharmacotherapy are observed with at least two different corticosteronesynthesis inhibitors and two different benzodiazepines.

Referring to FIG. 4, we see the results of experiments designed toinvestigate the effects of the combination of ineffective doses ofketoconazole (e.g., 12.5 mg/kg, ip) and alprazolam (e.g., 1 mg/kg, ip)on cocaine self-administration when different groups of rats weretrained to self-administer different doses of cocaine. This is importantfor the same reason as provided above in our studies with metyrapone andoxazepam. The numbers on the X-axis represent the three doses of cocainethat were self-administered. “Vehicle” shows the number of infusionsself-administered when the vehicle (5% emulphor in 0.9% saline) wasdelivered. “Keto 12.5” depicts the number of cocaine infusionsself-administered following the delivery of the ineffective doses ofketoconazole (i.e., 12.5 mg/kg, ip), while “Alp 1” represents the numberof cocaine infusions self-administered following the delivery of theineffective dose of alprazolam (i.e., 1 mg/kg, ip). “Keto/Alp”represents the number of cocaine infusions self-administered followingpretreatment with the combination of the ineffective doses ofketoconazole and alprazolam. Clearly, this combination significantlyreduced cocaine self-administration regardless of the dose of cocainethat was available for self-administration. This indicates that theeffects of the combination pharmacotherapy would not easily be overcomeby increasing the intake or dose of cocaine.

The Effect of Ketoconazole and Oxazepam on IntravenousSelf-Administration of Cocaine

These experiments were designed to determine the effects of acombination of ketoconazole and oxazepam on intravenous cocaineself-administration in rats responding under a multiple, alternatingschedule of food reinforcement and cocaine self-administration.Referring to FIG. 5, the solid bar “VEH”) depicts the number of cocaineinfusions self-administered following pretreatment with the vehicle (5%emulphor in 0.9% saline) for the treatment drugs. The open bar (“EXT”)shows the results of extinction when responding on the “active” leveronly resulted in infusions of saline. The striped bar (“OX”) and theshaded bar (“KETO”) represent self-administration following pretreatmentwith the low, ineffective doses (oxazepam 10 mg/kg, ip; ketoconazole12.5 mg/kg, ip) of oxazepam and ketoconazole alone. These doses did notsignificantly affect cocaine self-administration (or food-maintainedresponding) when administered alone. The small-striped bar (“COMBO”)depicts the number of cocaine infusions self-administered following thedelivery of the combination pharmacotherapy (i.e., an injectionconsisting of the ineffective doses of oxazepam and ketoconazole). Ascan be seen in FIG. 5, the combination pharmacotherapy consisting ofoxazepam and ketoconazole reduced cocaine self-administration to levelsseen when only saline was delivered when the active lever was pressedduring extinction. The combination pharmacotherapy reduced cocaineself-administration to extinction levels without affectingfood-maintained responding, suggesting that the combination was reducingthe motivation to seek cocaine without affecting responding ormotivation for another reinforcer (i.e., food). These data furtherdemonstrate that the effects of the combination pharamacotherapy areobserved with different corticosterone synthesis inhibitors anddifferent benzodiazepines.

The Effect of CP-154,526 and Oxazepam on Intravenous Self-Administrationof Cocaine

These experiments were designed to determine the effects of acombination of CP-154,526 and oxazepam on intravenous cocaineself-administration in rats responding under a multiple, alternatingschedule of food reinforcement and cocaine self-administration. Theresults are presented in FIG. 6 as the percentage of baseline infusionsunder the conditions tested. The white bar (“Ext”) shows the results ofextinction when responding on the “active” lever only resulted ininfusions of saline. The bar labeled “CP-high” depicts the number ofinfusions self-administered following pretreatment with the high dose ofCP-154,526 (10-80 mg/kg, ip), while the “OX-high” bar depicts the numberof cocaine infusions self-administered following pretreatment with thehigh dose of oxazepam (5-25 mg/kg, ip). Both CP-154,526 and oxazepamreduced cocaine self-administration without affecting food-maintainedresponding at these doses. The “CP-low” and “OX-low” bar representresponding following pretreatment with the low, ineffective doses(CP-154,526, 5-25 mg/kg, ip; oxazepam, 5-25 mg/kg, ip) of CP-154,526 andoxazepam alone. These doses did not significantly affect cocaineself-administration or food-maintained responding when administeredalone. The “COM-low” bar depicts the number of cocaine infusionsself-administered following the delivery of the combinationpharmacotherapy (i.e., an injection consisting of the ineffective dosesof CP-154,526 and oxazepam). As can been seen from FIG. 6, thecombination pharmacotherapy consisting of CP-154,526 and oxazepamreduced cocaine self-administration to levels seen when only saline wasdelivered when the active lever was pressed during extinction. Thecombination pharmacotherapy reduced cocaine self-administration toextinction levels without affecting food-maintained responding,suggesting that the combination was reducing the motivation to seekcocaine without responding or motivation for another reinforcer (i.e.,food). These data also demonstrate that the effects of the combinationpharmacotherapy are observed with the combination of a benzodiazepineand a CRH receptor antagonist.

The Effect of Metyrapone and Alprazolam on IntravenousSelf-Administration of Cocaine

These experiments were designed to determine the effects of acombination of metyrapone and alprazolam on intravenous cocaineself-administration in rats responding under a multiple, alternatingschedule of food reinforcement and cocaine self-administration.Referring to FIG. 7, the left-most bar (“Veh”) depicts the number ofcocaine infusions self-administered following pretreatment with thevehicle (5% emulphor in 0.9% saline) for the treatment drugs. The barlabeled “Ext” shows the results of extinction when responding on the“active” lever only resulted in infusions of saline. The “Met-H” barshows the number of cocaine infusions self-administered followingpretreatment with the high dose of metyrapone (25-175 mg/kg, ip), whilethe “ALP-H” bar depicts the number of cocaine infusionsself-administered following pretreatment with the high dose ofalprazolam (1-5 mg/kg, ip). Both metyrapone and alprazolam reducedcocaine self-administration without affecting food-maintained respondingat these doses. The “Met-L” and “ALP-L” bars represent respondingfollowing pretreatment with the low, ineffective doses (metyrapone,25-50 mg/kg, ip; alprazolam 0.5-2 mg/kg, ip) of metyrapone andalprazolam alone. These doses did not significantly affect cocaineself-administration or food-maintained responding when administeredalone. The “COMBO” bar depicts the number of cocaine infusionsself-administered following the delivery of the combinationpharmacotherapy (i.e., an injection consisting of the ineffective dosesof metyrapone and alprazolam). The combination pharmacotherapyconsisting of metyrapone and alprazolam reduced cocaineself-administration to levels seen when only saline was delivered whenthe active lever was pressed during extinction. The combinationpharmacotherapy reduced cocaine self-administration to extinction levelswithout affecting food-maintained responding suggesting that thecombination was reducing the motivation to seek cocaine withoutaffecting responding or motivation for another reinforcer (i.e., food).

The Effect of Muscimol and CP-154,526 on Intravenous Self-Administrationof Cocaine

These experiments were designed to determine the effects of acombination of CP-154,526 and muscimol on intravenous cocaineself-administration in rats responding under a multiple, alternatingschedule of food reinforcement and cocaine self-administration. Theresults are shown in FIG. 8. The bar labeled “Ext” depicts the resultsof extinction when responding on the “active” lever only resulted ininfusions of saline. The “Mus-high” bar shows the number of infusionsself-administered following pretreatment with the high dose of muscimol(1-4 mg/kg, ip), while the “CP-high” bar depicts the number of cocaineinfusions self-administered following pretreatment with the high dose ofCP-154,526 (10-80 mg/kg, ip). Both muscimol and CP-154,526 reducedcocaine self-administration at these doses without affectingfood-maintained responding. The “Mus-low” and “CP-low” bars representresponding following pretreatment with the low, ineffective doses ofmuscimol (0.5-2.0 mg/kg, ip) and CP-154,526 (5-25 mg/kg, ip) alone.These doses did not significantly affect cocaine self-administration offood-maintained responding when administered alone. The “COM-low” bardepicts the number of cocaine infusions self-administered following thedelivery of the combination pharmacotherapy (i.e., an injectionconsisting of the ineffective doses of muscimol and CP-154,526). As canbeen seen in FIG. 8, the combination pharmacotherapy consisting ofmuscimol and CP-154,526 reduced cocaine self-administration close tolevels seen when only saline was delivered when the active lever waspressed during extinction. The combination pharmacotherapy reducedcocaine self-administration close to extinction levels without affectingfood-maintained responding, suggesting that the combination reduced themotivation to seek cocaine without affecting responding or motivationfor another reinforcer (i.e., food).

The Effect of Muscimol and Metyrapone on Intravenous Self-Administrationof Cocaine

These experiments were designed to determine the effects of acombination of muscimol and metyrapone on intravenous cocaineself-administration in rats responding under a multiple, alternatingschedule of food reinforcement and cocaine self-administration. Theresults are shown in FIG. 9. The bar labeled “Ext” depicts the resultsof extinction when responding on the “active” lever only resulted ininfusions of saline. The “Mus-high” bar shows the number of infusionsself-administered following pretreatment with the high dose of muscimol(1-4 mg/kg, ip), while the “Met-high” bar depicts the number of cocaineinfusions self-administered following pretreatment with the high dose ofmetyrapone (25-175 mg/kg, ip). Both muscimol and metyrapone reducedcocaine self-administration at these doses without affectingfood-maintained responding. The “Mus-low” and “Met-low” bars representresponding following pretreatment with the low, ineffective doses ofmuscimol (0.5-2.0 mg/kg, ip) and metyrapone (25-50 mg/kg, ip) alone.These doses did not significantly affect cocaine self-administration offood-maintained responding when administered alone. The “COM-low” bardepicts the number of cocaine infusions self-administered following thedelivery of the combination pharmacotherapy (i.e., an injectionconsisting of the ineffective doses of muscimol and metyrapone). As canbeen seen in FIG. 9, the combination pharmacotherapy consisting ofmuscimol and metyrapone reduced cocaine self-administration close tolevels seen when only saline was delivered when the active lever waspressed during extinction. The combination pharmacotherapy reducedcocaine self-administration close to extinction levels without affectingfood-maintained responding, suggesting that the combination of aGABA_(A) receptor agonist and a corticosterone synthesis inhibitorreduced the motivation to seek cocaine without affecting responding ormotivation for another reinforcer (i.e., food).

The Effect of Chronic Injections of Metyrapone on the Cue-InducedReinstatement of Extinguished Cocaine-Seeking Behavior

These experiments were designed to determine the effects of the chronicadministration of metyrapone on the cue-induced reinstatement ofextinguished cocaine seeking in rats. We used a model of the relapse tococaine seeking. This is an important experiment since the combinationpharmacotherapy would be administered to cocaine addicts on a chronicbasis. Referring to FIG. 11, the bar labeled “SA” depicts the number ofresponses made on the “active” lever during cocaine self-administration.The bar labeled “EXT” depicts the number of responses on the “active”lever during extinction when responding on this lever only resulted ininfusions of saline. The bar labeled “VEH” represents responding on the“active” lever during reinstatement testing following pretreatment withthe vehicle (5% emulphor in 0.9% saline). The bar labeled “Metyrapone”depicts the number of responses on the “active” lever duringreinstatement testing following the chronic delivery of metyrapone (50mg/kg, ip, once per day for 14 days). As can be seen in FIG. 11, thechronic administration of metyrapone reduced cocaine seeking to levelsseen when only saline was delivered then the active lever was pressedduring extinction. These data demonstrate that metyrapone remainseffective in blocking the relapse of cocaine seeking following chronicadministration.

Effects of the Combination of CP-154,526 and Oxazepam on the Cue-InducedReinstatement of Extinguished Cocaine-Seeking Behavior

These experiments were designed to determine the effects of acombination of CP-154,526 and oxazepam on the cue-induced reinstatementof extinguished cocaine seeking in rats. Referring to FIG. 12, the setof bars labeled “Self-Admin” depict the number of responses made on the“active” lever and a second “inactive” lever during cocaineself-administration. The first two bars in the set represent theresponses of rats that eventually received the vehicle (5% emulphor in0.9% saline) as treatment drugs during reinstatement testing. The thirdand fourth bars depict the response of rats that eventually received thecombination pharmacotherapy (i.e., an injection consisting of theineffective doses of CP-154,526 and oxazepam as determined in thecocaine self-administration experiments) during reinstatement testing.The rats were only injected with the vehicle or the combinationpharmacotherapy once, which was 30 minutes before the start of thesession for reinstatement testing. The responses duringself-administration and extinction are only presented in FIG. 12 todemonstrate that there were no significant differences in respondingbetween the groups. Responding on the “inactive” lever produced noprogrammed consequences at any time. The second set of bars, labeled“Extinction”, depicts the number of responses on the “active” and“inactive” levers during extinction when responding on the “active”lever only resulted in infusions of saline. The third set of bars,labeled “Reinstatement”, depicts the number of responses on the “active”and “inactive” levers during reinstatement testing following thedelivery of the combination pharmacotherapy (i.e., an injectionconsisting of the ineffective doses of CP-154,526 and oxazepam asdetermined in the cocaine self-administration experiments). As can beclearly seen, the combination pharmacotherapy consisting of CP-154,526and oxazepam reduced cocaine seeking (i.e., responding on the activelever during reinstatement) to levels seen when only saline wasdelivered when the active lever was pressed during extinction. Thecombination pharmacotherapy reduced reinstatement (relapse) toextinction levels without affecting food-maintained responding. Thissuggests that the combination reduced the motivation to seek cocainewithout affecting responding or motivation for another reinforcer (i.e.,food).

No Evidence of Pharmacokinetic Interaction Between Cocaine, Metyrapone,and Oxazepam

Adult male Wistar rats (90 to 120 days old) were implanted with chronic,indwelling jugular catheters and were allowed to recover from surgery.On the test day, the rats were pretreated with intraperitonealinjections of various combinations of oxazepam and metyrapone (asindicated in the table of FIG. 14) or vehicle (5% emulphor in saline) 30minutes before the cocaine injections were administered. Theoxazepam/metyrapone combinations were selected from our behavioralstudies that demonstrated that these combinations reduced cocaineself-administration or the cue-induced reinstatement of extinguishedcocaine seeking without affecting food-maintained responding. Thirtyminutes following the drug combination or vehicle injection, the ratsreceived intravenous injections of cocaine (0.25 mg/kg/infusion) every 2minutes for 1 hour. After the final injection of cocaine, blood wascollected from the catheter for the analysis of cocaine and itsmetabolites ecgonine methyl ester and benzoylecgonine. Concentrations ofmetyrapone and metyrapol as well as oxazepam were also determined. Alldrug concentrations were determined using GCMS procedures. The resultsof these studies demonstrated that the combinations of oxazepam andmetyrapone had no effect on the plasma concentrations of cocaine or itsmetabolites. These studies also demonstrated that metyrapone andoxazepam did not influence plasma concentrations of each other.Furthermore, the presence of cocaine did not affect the plasmaconcentrations of metyrapone or oxazepam. These data suggest that thebehavioral effects we have observed in rats are not due topharmacokinetic interactions among the various drugs.

A Combination of Oxazepam and Metyrapone Tested in the Forced Swim Test,an Animal Model of Depression

The Forced Swim Test (FST) is an animal-model that possesses predictivevalidity for assessing a drug's anti-depressive efficacy. The subject isexposed to an inescapable, life-threatening situation to elicit learnedhelplessness. To achieve this, rats are placed in a cylinder filled withwater from which they cannot escape and in which they must swim to stayafloat. At a point in time when the rat ‘realizes’ its situation ishopeless, despair-like behavior appears and rather than attempting toescape or swim, the rat becomes immobile. The time in this immobilityposture is the behavior that is measured as despair. Oxazepam, abenzodiazepine, and metyrapone, an 11-β-hydroxylase inhibitor ofcorticosterone synthesis, have been shown to have anxiolytic andanti-depressant efficacy, respectively. The potential antidepressantproperties of oxazepam and metyrapone administered alone and togetherboth acutely and chronically were evaluated in male Wistar rats usingthe FST. Rats were injected with one of the drugs (5 or 10 mg/kgoxazepam, 25 or 50 mg/kg metyrapone) or combinations thereof both on dayone after testing and again on day two before testing (acute) or forfourteen days before initiating testing on day one (chronic). The acuteand chronic administrations of the drugs, alone and in combination, wereeffective in reducing immobility in the FST, indicating that thispharmacotherapy has antidepressant activity.

Learned helplessness is the construct on which the validity of using theFST as a model of depression is based. In humans, learned helplessnessis often manifested as a symptom of depression, which appears as a lossof coping ability. For that reason we believe that drugs that have theeffect of decreasing the time of immobility in the FST have potential ascandidates for lessening the loss of coping ability seen in the humanmodel of depression. In the current studies, oxazepam and metyraponewere tested alone and in combination in the FST to determine whetherthese agents might show antidepressant activity.

The parameters of the study were outlined above. More specifically, maleWistar rats from Harlan weighing 275-400 grams were used. The rats wereallowed to acclimate at least one day in the Animal Resources Facilityafter arrival before being tested. To perform the FST, a Plexiglascylinder (40 cm tall×18 cm diameter) was filled with fresh, 25° C. waterto a depth of 20 cm, which is deep enough so the rat cannot touchbottom, yet far enough from the rim to prevent the rat from escaping.Rats were injected intraperitoneally with either vehicle, drugs, orcombinations of oxazepam and metyrapone on day one after testing andagain on day two before testing (acute) or for fourteen days beforeinitiating testing on day one (chronic). On day one, the rat was removedfrom his cage, placed in the water, and observed for fifteen minutes.Generally, for the first few minutes, the rat would swim around with hispaws thrashing above the water line, sniff, dive, and attempt to jumpout of the cylinder. Such actions were deemed escape-oriented behavior.Following the escape-oriented behavior was a time characterized by therat discontinuing its attempts to escape. Generally, the rat wouldeither tread water, exerting only enough energy to keep its head abovewater, or would float with only its nose above the water line. Thissecond phase of behavior was deemed the immobility posture. Length oftime spent in escape-oriented behavior and immobility posture wasrecorded. Then the rat was removed from the water, dried with a towel,and returned to his home cage. On day two the procedure was repeated forfive minutes and the time spent engaging in escape-oriented behavior andimmobility posture were recorded. The second day's duration ofimmobility was compared among the different groups. Dosage groups werecompared to the vehicle-injected controls using a one way ANOVA withp<0.05. If the Immobility Time for a drug group was statisticallysignificant compared to that of the vehicle group, the drug combinationwas considered to exhibit an antidepressant-like effect.

The effects of the chronic administration of oxazepam and metyraponewere more profound in the combination-treated groups. Only one group towhich individual drugs were administered, the Met50 group, showed alessening of immobility time. This is suggestive of a synergistic actionwhen both drugs are administered simultaneously. Perhaps thissynergistic effect can be explained by an increase in oxazepam'sagonistic action on the GABA_(A) receptor induced by the metabolicby-products of metyrapone. When metyrapone inhibits corticosteronesynthesis, the concentrations of two precursors upstream ofcorticosterone, 11-Deoxycorticosterone (11-DOC) and Progesterone (Prog),increase. This increase may shunt the pathway towards the production ofGABA_(A)-active neurosteroids such as allopregnanolone andtetrahydrodeoxycorticosterone. These two neurosteroids bindallosterically to the GABA_(A) receptor resulting in an increase ofCl-flowing into the cell, thus causing hyperpolarization and decreasedneuronal excitability. The possible outcome is that both oxazepam (bydirect binding) and metyrapone (indirectly through neurosteroids) bothinfluence GABA_(A) currents via allosteric mechanisms. Regardless of themechanism of action, it is clear that the combination of Ox10/Met50elicited the largest reduction in immobility time. Tolerance appears tohave formed in the chronically treated groups, especially to thosegroups who received only Ox or Met. This is evident by the observationthat the means for these groups were equal to or exceeded the vehicle.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is: 1-38. (canceled)
 39. A pharmaceutical compositionfor the treatment of addiction comprising a first agent that targets thehypothalamo-pituitary-adrenal (HPA) axis and down-regulates the effectof cortisol in a subject to whom the composition is administered, and asecond agent selected from the group consisting of: a benzodiazepine,mirtazapine, gabapentin, muscimol, baclofen, progabide, riluzole,vigabatrin, tiagabine, lamotrigine, carbamazepine, and topiramate, orpharmaceutically acceptable salts, solvates, hydrates, prodrugs,structural analogs or polymorphs thereof.
 40. The pharmaceuticalcomposition of claim 39, wherein the first agent is a chemical compoundor a pharmaceutically acceptable salt thereof.
 41. The pharmaceuticalcomposition of claim 40, wherein the first agent is metyrapone or asalt, solvate, hydrate, prodrug, structural analog, or polymorphthereof.
 42. The pharmaceutical composition of claim 41, wherein thesecond agent is a benzodiazepine.
 43. The pharmaceutical composition ofclaim 42, wherein the benzodiazepine is oxazepam or chlordiazepoxide.44. The pharmaceutical composition of claim 39, wherein the second agentis benzodiazepine or a salt thereof.
 45. The pharmaceutical compositionof claim 44, wherein the benzodiazepine is oxazepam or chlordiazepoxide.46. The pharmaceutical composition of claim 39, wherein the second agentis mirtazapine or a salt thereof.
 47. The pharmaceutical composition ofclaim 39, wherein the second agent is gabapentin or a salt thereof. 48.The pharmaceutical composition of claim 39, wherein the second agent ismuscimol progabide, riluzole, baclofen, vigabatrin, tiagabine,lamotrigine, carbamazepine, topiramate or baclofen or pharmaceuticallyacceptable salts thereof.
 49. The pharmaceutical composition of claim39, comprising about 150-1000 mg of said first agent and about 5-100 mgof said second agent.
 50. The pharmaceutical composition of claim 49,wherein said composition comprises: (i) about 90 mg of said first agentand about 4 mg of said second agent; (ii) about 180 mg of said firstagent and about 8 mg of said second agent; or (iii) about 360 mg of saidfirst agent and about 16 mg of said second agent.
 51. The pharmaceuticalcomposition of claim 50, wherein said first agent comprises metyraponeand said second agent comprises a benzodiazepine.
 52. The pharmaceuticalcomposition of claim 51, wherein said second agent is oxazepam.
 53. Thepharmaceutical composition of claim 52, wherein said composition is inunit dosage form.
 54. The pharmaceutical composition of claim 49,comprising about 250-1000 mg of said first agent and about 5-60 mg ofsaid second agent.
 55. The pharmaceutical composition of claim 39,further comprising a third agent that inhibits activity in thesympathetic nervous system.
 56. The pharmaceutical composition of claim39, wherein the ratio of the first agent to the second agent is about100:1, 90:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1,30:1, 25:1, 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, orabout 1:1.
 57. The pharmaceutical composition of claim 48, wherein theratio of the first agent to the second agent is about 100:1, 90:1, 80:1,75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1,15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or about 1:1. 58.The pharmaceutical composition of claim 51, wherein the dosage ofmetyrapone is about 25-1500 mg, 50-1250 mg, 100-1250 mg, 100-1000 mg,250-1000 mg, 500-1000 mg or 750-1000 mg; and wherein dosage of saidsecond agent is about 5-50 mg, about 5-40 mg, about 5-30 mg, about 5-20mg, about 5-10 mg, about 10-50 mg, about 10-40 mg, about 10-30 mg, about20-50 mg, about 20-40 mg, about 20-30 mg, about 30-50 mg or about 30-40mg.
 59. The pharmaceutical composition of claim 58, wherein the dosageof metyrapone is about 5, 10, 16, 25, 50, 90, 100, 180, 200, 250, 300,360, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000, 1250,or 1500 mg; and wherein dosage of said second agent is about 1, 4, 5, 8,10, 16, 20, 25, 30, 35, 40, 45, 50 mg.
 60. The pharmaceuticalcomposition of claim 57, wherein the dosage of metyrapone is about 5,10, 16, 25, 50, 90, 100, 180, 200, 250, 300, 360, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 1000, 1250, or 1500 mg; and whereindosage of oxazepam is about 1, 4, 5, 8, 10, 16, 20, 25, 30, 35, 40, 45,50 mg.
 61. The pharmaceutical composition of claim 53, wherein thecomposition consists essentially of: (i) about 90 mg of metyrapone andabout 4 mg of oxazepam; (ii) about 180 mg of metyrapone and about 8 mgof oxazepam; or (iii) about 360 mg of metyrapone and about 16 mg ofoxazepam.
 62. The pharmaceutical composition of claim 61, wherein saidcomposition is in unit dosage form.
 63. The pharmaceutical compositionof claim 53, wherein the composition consists of: (i) about 90 mg ofmetyrapone and about 4 mg of oxazepam; (ii) about 180 mg of metyraponeand about 8 mg of oxazepam; or (iii) about 360 mg of metyrapone andabout 16 mg of oxazepam.
 64. The pharmaceutical composition of claim 63,wherein said composition is in unit dosage form.